Aminosterol compounds useful as inhibitors of the sodium/proton exchanger (NHE)

ABSTRACT

Aminosterol compounds are described that are useful as inhibitors of the sodium/proton exchanger (NHE). Methods of using such aminosterols compounds are also enclosed, including those employing compounds that are inhibitors of a spectrum of NHEs as well as those using compounds that are inhibitors of only one specific NHE. Advantageous screening techniques and assays for evaluating a compound&#39;s therapeutic activity are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to U.S. Ser. No. 08/416,883, now pendingwhich is the U.S. national phase of International Application No.PCT/US94/10265, filed Sep. 13, 1994.

FIELD OF THE INVENTION

The present invention relates to aminosterol compounds useful asinhibitors of the sodium/proton exchanger (NHE). The invention is alsodirected to pharmaceutical compositions containing such compounds, andthe use of such compounds for inhibiting NHE. The invention is furtherdirected to assaying techniques for screening compounds for theirefficacy as NHE inhibitors.

BACKGROUND OF THE INVENTION

Each of the body's cells must maintain its acid-base balance or, morespecifically, its hydrogen ion or proton concentration. Only slightchanges in hydrogen ion concentration cause marked alterations in therates of chemical reactions in the cells--some being depressed andothers accelerated. In very broad and general terms, when a person has ahigh concentration of hydrogen ions (acidosis), that person is likely todie in a coma, and when a person has a low concentration of hydrogenions (alkalosis), he or she may die of tetany or convulsions. In betweenthese extremes is a tremendous range of diseases and conditions thatdepend on the cells involved and level of hydrogen ion concentrationexperienced. Thus, the regulation of hydrogen ion concentration is oneof the most important aspects of homeostasis.

A shorthand method of expressing hydrogen ion concentration is pH:pH=log 1/(H concentration)=-log (H⁺ concentration). The normal cell pHis 7.4, but a person can only live a few hours with a pH of less than7.0 or more than 7.7. Thus, the maintenance of pH is critical forsurvival.

There are several mechanisms of maintaining pH balance. For example,during quiescence and constitutive growth, cells appear to utilize thechloride/bicarbonate exchanger, a well-studied device which provides forproton exchange across cells such as the red cell.

In addition, during accelerated periods of growth, which are induced bymitogens, growth factors, sperm, etc., cells engage another piece ofcellular equipment to handle the impending metabolic burst. This is thesodium/proton (Na⁺ /H⁺) exchanger--the "NHE," which is also called an"antiporter." Because the NHE functions in a number of roles and in anumber of tissues, the body has developed a family of NHEs, and recentwork has elucidated a family of NHE "isoforms" that are localized incertain tissues and associated with various functions. The NHE isoformslisted below are most likely to be significant.

NHE1 is a housekeeping exchanger and is believed to be unregulated inhypertension. It is thought to play a role in intracellular pH conduct.Also, it is believed that control of this exchanger will protect apatient from ischemic injury.

NHE1 is associated genetically with diabetes and, thus, inhibition mightalter evolution of diabetes through effects on beta cells in thepancreas. In addition, vascular smooth muscle proliferation, responsiveto glucose, is associated with increased expression of NHE1a.

NHE1β is present on nucleated erythrocytes. It is inhibited by highconcentrations of amiloride. This NHE isoform is regulated by adrenergicagents in a cAMP-dependent fashion.

NHE2 is associated with numerous cells of the GI tract and skeletalmuscle. Inhibition could alter growth of hyperplastic states orhypertrophic states, such as vascular smooth muscle hypertrophy orcardiac hypertrophy. Cancers of muscle origin such as rhabdomyosarcomaand leiomyoma are reasonable therapeutic targets.

NHE3 is associated with the colon. The work described below shows it tobe associated with endothelial cells. Inhibition would affect functionssuch as water exchange in the colon (increase bowel fluid flux, which isthe basis of, e.g., constipation), colonic cancer, etc. On endothelialcells, normal growth would be inhibited through inhibition of theexchanger.

NHE4 is associated with certain cells of the kidney. It appears to playa role in cellular volume regulation. Specific inhibitors might affectkidney function, and hence provide therapeutic benefit in hypertension.

NHE5 is associated with lymphoid tissue and cells of the brain.Inhibition of NHE5 should cause inhibition of proliferative disordersinvolving these cells. NHE5 is a likely candidate for the proliferationof glial cells in response to HIV and other viral infections.

As indicated by the above, although the NHE functions to assist thebody, the inhibition of NHE function should provide tremendoustherapeutic advantages. For example, although the NHE normally operatesonly when intracellular pH drops below a certain level of acidity, upongrowth factor stimulation the cell's NHEs are turned on even though thecell is poised at a "normal" resting pH. As a consequence, the NHEsbegin to pump protons from the cell at a pH at which they would normallybe inactive. The cell undergoes a progressive loss of protons,increasing its net buffering capacity or, in some cases, actuallyalkalinizing. In settings where the pump is prevented from operating,the growth stimulus does not result in a cellular effect. Thus,inhibitors of the NHE family are likely to exert growth-inhibitoryeffects.

During severe acid stress--the condition that a tissue might find itselfin when deprived of oxygen (or a blood supply)--the NHE family isbelieved to contribute to subsequent irreversible damage. For example,when blood flow to the heart is impaired, local acidosis occurs. Heartmuscle cells develop a profound internal acidity. The acidity, in turn,activates otherwise dormant NHEs. These exchangers readily eliminateprotons from the cell, but in exchange for sodium. As a consequence,intracellular sodium concentrations rise. Subsequently, thesodium-calcium exchanger is activated, exchanging internal sodium forexternal calcium. The rise in internal Ca⁺ concentrations leads to celldeath, decreased contractility, and arrhythmias. Thus, post ischemicmyocardial damage and associated arrhythmias are believed to arise froman NHE-dependent mechanism, and inhibition of this NHE should thereforeprevent such occurrences. If the NHE inhibited the internalization ofNa⁺ and slowed down metabolic activity as a consequence of the depressedpH, damage of the cell could be avoided. Hence, there is an interest inthe development of NHE inhibitors for use in cardiac ischemia.

Other members of the NHE family appear to play a more classical role inwater and sodium transport across epithelial surfaces. Specifically, theNHE3 isoform found in the colon is believed to play a role in regulatingthe fluid content of the colonic lumen. This pump is inhibited in casesof diarrhea. The NHE3 isoform present on the proximal tubules of thekidney is believed to play a similar role with respect to renal salt andacid exchange. Accordingly, inhibitors of the NHE family have beenregarded as therapeutic modalities for the treatment of hypertension.

In view of the expected value of the inhibition of NHE action,scientists have sought out NHE inhibitors. The most widely studiedinhibitor of NHE is amiloride, a guanidine-modified pyrazine usedclinically as a diuretic. A number of derivatives have been generated,incorporating various alkyl substitutions. These derivatives have beenstudied with the several isoforms of NHE that are known and describedabove, except for NHE5, for which there is no known inhibitor.

The activities of these inhibitors against these specific exchangershave been previously determined. As seen in Table A below, eachexchanger exhibits a different spectrum of response to each inhibitor:

                  TABLE A                                                         ______________________________________                                               Amiloride    DMA     MPA                                                      K.sub.i (μM)                                                                            K.sub.i (μM)                                                                       K.sub.i (μM)                                   ______________________________________                                        NHE1     3              0.1     0.08                                          NHE2     3              0.7     5.0                                           NHE3     100            11      10                                            ______________________________________                                         Notes:                                                                        DMA = dimethylamiloride; MPA = methylpropylamiloride.                         See Counillon et al., Molecular Pharmacology 44, 1993, 1041-1045.        

The NHE inhibitors described by Counillon et al. exhibit specificity forNHE1. They therefore serve a therapeutic value in the treatment ofconditions where inhibition of this isoform is beneficial. However,these inhibitors do not target the other known NHE isoforms--e.g., NHE3is unaffected.

NHE3, as is demonstrated below, is expressed on endothelial cells, andits inhibition results in anti-angiogenic effects. The spectrum of NHEisoforms inhibited by the aminosterol compounds in accordance with theinvention are different from those inhibited by the amiloride or theCounillon et al. compounds, and have different, distinct pharmacologicaleffects.

In addition, Counillon et al. also reported that certainbenzoylguanidine derivatives inhibit other NHE isoforms. In particular,(3-methylsulfonyl-4-piperidinobenzoyl) guanidine methanesulfonateexhibits particular selectivity to the NHE1 as shown in the table below.

                  TABLE B                                                         ______________________________________                                               NHE Isoform                                                                            K.sub.i (μM)                                               ______________________________________                                               NHE1     0.16                                                                 NHE2     5.0                                                                  NHE3     650                                                           ______________________________________                                    

These benzoylguanadine compounds, which are based on the chemicalstructure of amiloride, exhibit greatest specificity for inhibiting NHE1while retaining considerable activity against NHE2 and NHE3. To achievepharmacological inhibition of NHE1, the widely distributed"housekeeping" isoform, undesirable inactivation of NHE2 and NHE3 wouldoccur.

Those in the art have therefore continued to search for NHE inhibitorsthat exhibit selective action against a single, specific NHE. Suchinhibitors would permit more precise inhibition of a tissue byperturbing the effect of the NHE on its growth.

Thus, artisans have recognized that the development of variousNHE-specific inhibitors would allow for the development of new therapiesfor a whole host of diseases or conditions, including: treatingarrhythmias; treating and preventing cardiac infarction; treating andpreventing angina pectoris and ischemic disorders of the heart; treatingand preventing ischemic disorders of the peripheral and central nervoussystem; treating and preventing ischemic disorders of peripheral organsand limbs; treating shock; providing anti-arteriosclerotic agents;treating diabetic complications; treating cancers; treating fibroticdiseases, including fibroses of lung, liver and kidney; and treatingprostatic hyperplasia. Other therapeutic targets include: treatment ofviral disease, such as HIV, HPV and HSV; prevention of malignancies;prevention of diabetes (i.e., islet cell injury); prevention of vascularcomplications of diabetes; treatment of disorders of abnormalneovascularization, e.g., macular degeneration, rheumatoid arthritis,psoriasis, cancer, malignant hemangiomas; prevention of vascularretenosis; prevention of hypertension-associated vascular damage;immunosuppression; and treatment of collagen vascular disorders.

Inhibitors of NHEs of bacteria fungi and protozoa would also be valuableas specific antimicrobials. It is known that all living cells use an NHEof one form or another to maintain intracellular Na⁺ and pH homeostasis.NHEs have been cloned from numerous bacteria and fungi, and bear somesequence homology to the mammalian isoforms. Using a highly specificbacterial or fungal NHE as a target, it should be possible to develop ahighly specific inhibitor of such an exchanger, one that is particularlyadvantageous or that lacks activity against the mammalian isoforms. Suchcompounds would be useful as antibiotics of a different mechanism.

Thus, there is a need in the art for specific inhibitors of NHEs. Thereis further a need to develop NHE inhibitors for various therapeuticuses.

SUMMARY OF THE INVENTION

The present invention fulfills needs felt in the art by providingvarious aminosterol compounds that inhibit various NHEs. The inventionis directed to aminosterol compounds that exhibit inhibitory action onNHEs, and to compositions containing such compounds.

Thus, the invention is directed to newly isolated and synthesizedaminosterol compounds that are useful as inhibitors of NHEs, such ascompounds FX1A, FX1B, 1360, 1361, 371, 1437, and 353. Some of thesesteroid compounds have been found to inhibit a spectrum of NHEs, whileothers have been found to advantageously inhibit a single, specific NHE.

Further, the invention is directed to pharmaceutical uses and therapiesemploying the compounds of the invention. The invention is also directedto new uses for squalamine, which had been previously isolated andcharacterized.

Additionally, the invention is directed to advantageous screeningmethods for evaluating a compound's therapeutic efficacy. In particular,a tadpole assay has been developed, which has been found to be aconvenient tool for screening compounds for NHE inhibition andtherapeutic effects.

An especially preferred compound of the invention is compound 1436 (or apharmaceutically acceptable salt thereof). The invention is directed toa pharmaceutical composition comprising an effective amount of thiscompound and a pharmaceutically acceptable vehicle or carrier. Theinvention is further directed to a method of inhibiting theproliferation of cells, comprising administering an effective amount ofcompound 1436, and particularly to such a method where the cells aremalignant cells, vascular smooth muscle cells, bronchial smooth musclecells, fibroblasts, lymphocytes or lymphoid tissue, muscle, bone,cartilage, epithelium, hematopoietic tissue, or neural tissue.Furthermore, the invention is directed to a method of inhibiting theproliferation of cells, comprising administering an effective amount ofa combination comprising compound 1436 and squalamine. The inventionalso relates to a method for suppressing the immune system by inhibitingthe proliferation of lymphocytes, comprising administering an effectiveamount of compound 1436. In addition, the invention involves suppressingthe growth of a vertebrate, comprising administering an effective amountof compound 1436. The invention also relates to treating a viralinfection by suppressing the growth of a viral target cell, comprisingadministering an effective amount of compound 1436. A method ofcontrolling arterial pressure, comprising administering an effectiveamount of compound 1436, is also preferred. Also, the invention isdirected to a method of protecting against cardiac ischemia, comprisingadministering an effective amount of compound 1436. The invention alsorelates to a method of preserving transplanted organs, comprising theadministration of an effective amount of compound 1436. Furthermore, theinvention is directed to a method of treating an infection caused by anmicrobial agent, such as bacteria, viruses, fungi, and protozoa,comprising the administration of an effective amount of compound 1436.The invention also pertains to the administration of an effective amountof this compound to inhibit an NHE.

The invention is also directed to a method of inhibiting NHE3,preferably to a method of specifically inhibiting this NHE isoform thatis expressed in a pathological process, comprising the administration ofan effective amount of squalamine (or its pharmaceutically acceptablesalt). Another method according to the invention involves inhibiting thegrowth of endothelial cells, especially ones of new capillaries,comprising administering an effective amount of squalamine.

The invention also pertains to a or method for evaluating a compound forNHE-inhibiting activity or anti-angiogenic activity, comprisingperforming a tadpole assay comprising the steps of: (i) preparing anaqueous solution containing a compound to be assayed (e.g., at aconcentration of 10 μg/ml); (ii) introducing a tadpole into thesolution; and (iii) after at least one interval of time (e.g., about anhour), observing the tadpole (e.g., its tail and/or hands and feet)under a microscope. Preferably, the tadpole is a Xenopus tadpole, morepreferably a stage 59-60 Xenopus tadpole. Such an assay can be usedalone or in combination with another assay, e.g., a chickchorioallantoic membrane assay and/or a chick embryo vitelline capillaryregression assay.

Other aspects, objects and advantages will be apparent from the detaileddisclosure below, which illustrates preferred features and embodimentsof the invention in conjunction with the appended drawing figures.

BRIEF DESCRIPTION OF DRAWING FIGURES

FIGS. 1A and 1B show the inhibition of rabbit sodium/proton exchangerisoform 3 (NHE3) by squalamine. FIG. 1A is a plot of the rate of pHrecovery (y-axis) as a function of restored extracellular sodium ionconcentration (x-axis) for cells acidpreloaded by exposure to 40 mM NH₄Cl, with the curve marked by "+" being for control (no drug) and thecurve marked by "Δ" being for squalamine. FIG. 1B shows the actualinternal pH (y-axis) as a function of time (x-axis) following additionof 5 μg/ml of squalamine for cells not acid-preloaded.

FIG. 2A shows the lack of inhibition of rabbit sodium/proton exchangerisoform 1 (NHE1) by squalamine. FIG. 2B shows the lack of inhibition ofhuman NHE1 by squalamine. In these plots of internal pH vs. time, thecurve marked by "o" is for squalamine and that marked by "+" is thecontrol (cells incubated in the absence of squalamine).

FIGS. 3A, 3B and 3C illustrate that endothelial cells exhibit greatersensitivity to squalamine (bar above 3 on the x-axis) than to othermembrane-active agents, and that endothelial cells are more sensitive tosqualamine than are epithelial cells and fibroblasts. FIG. 3A is for theadministration of 1 μg/ml of the agent against bovine pulmonaryendothelial cells, whereas FIGS. 3B and 3C are for administration of 10μg/ml of the membrane-active agents to human epithelial cells and tohuman foreskin fibroblasts, respectively.

FIGS. 4A, 4A-1, 4B, 4B-1, 4C, and 4C-1 show the suppression of thegrowth of murine melanoma, respectively through the subcutaneous,intraperitoneal and oral administration of squalamine.

FIG. 5 demonstrates the suppression of the growth of human melanoma1205Lu in immunocompromised (RAG-1) mice by squalamine at variousdosages ("o"=10 mg/kg/d, "+"=20 mg/kg/d, "" =40 mg/kg/d; d=day).

FIG. 6 illustrates the suppression of murine melanoma in mice byintraperitoneal administration of compound 319.

FIG. 7 shows the pharmacokinetic clearance of compound 319 from a mouseIV PK study.

FIG. 8 shows the pharmacokinetic clearance of squalamine from a mouse IVPK study.

FIG. 9 is an HPLC profile for aminosterols derived from the liver of thedogfish shark, illustrating the diversity of these compounds.

FIG. 10 illustrates the inhibitory effect of compound 1436 on NHE3.

FIG. 11 illustrates the effect of compound 1436 on survival in micebearing L1210 leukemia.

FIG. 12 illustrates that squalamine and compound 1436 exhibit synergy insuppressing growth of murine melanoma in mice.

FIGS. 13 and 14 show the in vitro suppression of the growth of humancoronary artery smooth muscle by compound 1436 (FIG. 13) and squalamine(FIG. 14), with absorbance plotted vs. concentration in μg/ml.

FIG. 15 is a expanded plot of the data shown in FIGS. 13 and 14,evidencing that both compound 1436 and squalamine suppress in vitro thegrowth of human coronary artery smooth muscle.

FIG. 16 illustrates that compound 1436 suppresses the growth of mice ina dose-dependent fashion.

FIGS. 17A and 17B show the effect of compound 353 versus squalamine onhuman melanoma.

DETAILED DESCRIPTION OF THE INVENTION

Syntheses of Aminosterol Compounds

The steroid known as squalamine is the subject of U.S. Pat. No.5,192,756 to Zasloff et al., the disclosure of which is hereinincorporated by reference. This compound is a broad-spectrum antibiotic,killing bacteria, fungi and protozoa. The absolute stereochemistry forsqualamine, compound 1256, is shown below. The total chemical synthesisof squalamine was reported in 1994.

EXAMPLE 1 Preparation of Shark Liver Isolates

In addition to squalamine, at least ten other distinctly differentaminosterols have been recovered from extracts of dogfish shark liver.To prepare the aminosterols, shark liver was extracted inmethanol:acetic acid. The aqueous extract was adsorbed to C18 silica andeluted with 70% acetonitrile, and the eluate was adsorbed to SP-sephadexand eluted with 1.5M NaCl. The eluate was adjusted to 5M NaCl, and thesteroids salted out. The precipitate was filtered over Celite and elutedwith hot water, followed by methanol. The eluate was reduced in volumeand applied to a 1-inch C18 column, and subjected to chromatographyutilizing an increasing gradient in acetonitrile. Fractions werecollected, concentrated by evaporation, and analyzed separately by thinlayer chromatography (TLC).

The HPLC profile of the aminosterols isolated from 40 kg of shark liveris shown in FIG. 9. Final HPLC purification was performed as describedin Moore et al., Proc. Natl. Acad. Sci. 90, 1993, 1354-1358. HPLCfractions were resolved individually by silica thin layer chromatography(6:3:1 CH₂ Cl₂ :MeOH:NH₄ OH) visualized in iodine vapor. Fraction 40represents the more hydrophilic portion of the elution profile, andfraction 66 represents the more hydrophobic portion.

Squalamine elutes beginning at about fraction 62 and continues untilfraction 80. In addition, other steroids can be seen eluting betweenfractions 43-47 (R_(f) 82), 53-55 (R_(f) 1.02), 56-59 (R_(f) 0.51),57-62 (R_(f) 0.96), 60-64 (R_(f) 0.47) and 61-66 (R_(f) 1.06), asdescribed below in Table 1.

                  TABLE 1                                                         ______________________________________                                        Chemical and Structural Characterization of                                   Aminosterols Isolated From Shark Liver                                        FRACTION  TLC Rf      Compound No.                                                                             Mass                                         ______________________________________                                        43-47     0.82        FX 1A      664.5                                                              FX 1B      641.5                                        53-55     1.02        1360       641.49                                       56-59     0.51        FX 3                                                    57-62     0.96        1437       657.52                                       60-64     0.47        1436       684.52                                       61-66     1.05        1361       543.48                                       63-80     1.0         1256       627.98                                       ______________________________________                                    

The structures for some of these compounds are shown below. ##STR1##

Each of these entities was isolated, purified, characterized, and thestructure determined by NMR as described below.

Compound 1360

This compound, the major steroid in Fraction 2 from preparative C18RP-HPLC, was purified by strong cation exchange usingsulfoethylaspartamide HPLC eluted with an increasing NaCl gradient.Steroid fractions were assayed by silica TLC developed in CH₂ Cl₂ :CH₃OH:NH₄ OH (6:3:1) and visualized with iodine. Steroid-fractions werethen pooled and re-chromatographed by C18 RP-HPLC with a gradient ofincreasing CH₃ CN in aqueous 0.1% TFA. TLC analysis of the purifiedcompound showed a single spot with R_(f) =1.02 with respect tosqualamine (R_(f) squalamine=1.0).

For subsequent isolations of compound 1360, strong cation exchangechromatography was not performed. Instead, pooled fractions frompreparative C18 RP-HPLC were subjected directly to C18 or C8 RP-HPLCusing a shallower CH₃ CN gradient. Fractions were assayed both by TLCand also by ¹ H-NMR for samples redissolved in D₂ O.

When analyzed by FAB-MS, the compound typically showed only a very weak(M+H)+ signal at 642.5 and an intense fragment at 562.5 daltons in thepositive ion mode. In negative ion FAB-MS, (M-H)- was observed at 640.4.Subsequent electrospray ionization (ESI-MS) analysis exhibited strong(M+H)+ and (M-H)- signals consistent with a parent mass of 641.4, alongwith many TFA adducts. This suggested that lability of sulfate incompound 1360 was more pronounced under FAB conditions.

Since the parent ion in FAB positive ion mode was very weak inintensity, accurate mass determination was conducted on the dessulfatefragment. An accurate mass of 561.49325 was observed. An accurate parentmass of 641.49 daltons was then calculated by adding the mass of SO₄ -,which matches with the molecular formula C₃₄ H₆₃ N₃ O₆ S. This molecularformula, with one additional oxygen and two fewer hydrogens whencompared to squalamine (C₃₄ H₆₅ N₃ O₅ S), was suggestive of a compoundbearing a carbonyl moiety.

¹ H-NMR of compound 1360 in D₂ O (300 MHz) revealed several featuresdistinguishing it from squalamine. For compound 1360, the resonanceappearing furthest downfield at δ=4.15 ppm showed a splitting pattern ofat least 7 signals with an integration of two protons; for squalamine,the most deshielded resonance at δ=4.2 ppm was the sulfate position(H24), resolved at 300 MHz as a multiplet of 5 signals with anintegration of one proton. At 2.6 ppm, compound 1360 also showed apoorly resolved multiplet, attributed to 2 protons. It was suspectedthat these resonances could be attributed to methylene protons alpha toa carbonyl, based on comparison to the literature. In squalamine theregion from 2.2-2.75 exhibited no resonances. Compound 1360 showed twodistinct methyl doublets, one at 0.95 ppm and the other at 1.1 ppm; thiscontrasts with squalamine, where three methyl groups split as doubletsoverlapping in the 0.9-1.05 ppm region. Other resonance characteristicswere quite similar for the two steroids. In the upfield methyl region,both compound 1360 and squalamine showed annular singlet methyl signalsat 0.85 and 0.65 ppm, positions 19 and 18 respectively. The resonancesfrom the steroid nucleus (1.0-2.1 ppm) and from spermidine also showedthe same characteristic pattern for both compound 1360 and squalamine.H7, at the alcohol position in the ring, resonates at 3.85 ppm for bothsteroids in D₂ O

A COSY (correlated spectroscopy) spectrum conducted in D₂ O (300 MHz)established the sulfate at position 27, --CH₂ OSO₃ --. Diagnosticcrosspeak patterns in the 2-dimensional frequency (2D) plot indicatedthat the doublet peak at δ=1.1 ppm was methyl 26, which then connectedto H25 at 3.05 ppm (hidden under the polyamine resonances), which inturn was the nearest neighbor to the complex multiplet at 4.15 ppm.

The polyamine region in the 2D map was consistent with the splittingpattern of spermidine, confirming that compound 1360 had the samepolyamine as squalamine. Otherwise, the 2D map in D₂ O was deficient inmany crosspeak signals, particularly in the steroid nucleus region, andcould not be used to established the complete primary structure.

Compound 1360 was dried in vacuo and dissolved in DMSO-d6 undernitrogen, and then sealed under nitrogen using cycles offreeze-thaw-pump. Both ID and 2D ¹ H-NMR spectra were conducted at 300MHz and at 600 MHz. All of the proton resonances of the compound in DMSOwere sharp (except for the polyamine region (2.8-3.1)) and shifted withrespect to assignments in D₂ O. For example, the multiplet for position27 was shifted upfield to 3.77 ppm and the H7 proton shifted to 3.60ppm. In addition, a new doublet signal (integration=1 H) appeared at4.17 ppm. This new resonance was identified as the hydroxyl at position7, based on its 2D connectivity to H7, which was unambiguous at 600 MHz.This resonance could not be observed in D₂ O due to its rapid exchangewith solvent. The 2D map of compound 1360 in DMSO reconfirmed thelocation of the sulfate group at position 27, which was first deducedfrom the 2D COSY map in D₂ O.

A careful comparison of 2D COSY maps for compound 1360 and squalaminesuggested the presence of a carbonyl at position 24. The multipletcentered at 2.5 ppm (2.6 ppm in D₂ O) with integration of 2 protons gavestrong crosspeaks that identified these resonances as H23a,b (locatedalpha to a carbonyl at position 24) and with nearest neighborconnections to H22a,b. In a total correlation spectroscopy experiment(TOCSY), a new crosspeak was discernible as H22/H21, due to propagationof magnetization along the tail of the steroid. Noticeably absent fromthe 2D COSY and TOCSY maps were signals that would allow propagation ofthe magnetization from positions 23 to 24 and then from 24 to 25. Unlikethe COSY of squalamine, which shows nearest neighbor crosspeaks for22-23-24-25-26/27, compound 1360 showed an interruption in connectivity,suggesting that a functional group at position 24 blocked transfer ofthe proton signal. The structure can be verified by reducing C═O to analcohol, however, since an alcoholic group at position 24 would allowfor complete proton connectivity from positions 21-26 and 27.

¹³ C-NMR of compound 1360 in DMSO indicated 34 carbon signals. Incomparison to squalamine, compound 1360 has one carbonyl at 212 ppm(C24). C27 resonated at 67 ppm, which is in good agreement with valuesreported for scymnol sulfate.

Compound 1361

Compound 1361 was isolated from shark liver preparations in twodifferent ways: first, as a degradation product of compound 1360; and,subsequently, as a minor aminosterol component fractionating withslightly faster retention time than squalamine during preparative C18RP-HPLC (component of Fraction VI). In early attempts to purify eachaminosterol to homogeneity, pooled fractions from C18 RP-HPLC weresubjected to silica gel flash chromatography with CH₂ Cl₂ :CH₃ OH:NH₄ OH6:3:1, and the aminosterols were assayed on TLC plates. The pools offree base steroids were then re-subjected to C18 RP-HPLC, using ananalytical column. The RP-HPLC elution profile showed two majorsteroids--compound 1360 and a second, more hydrophobic steroid flutingat higher % CH₃ CN. The lability of the sulfate at position 27 incompound 1360 led to the formation of compound 1361, apparently throughbase-catalyzed elimination.

Hallmark features of the ¹ H spectrum for compound 1361 in D₂ O (400MHz) included the absence of multiplet at δ=4.15 ppm corresponding tomethylene protons at position 27, bearing the sulfate. Two new singletprotons at δ=5.95 and 6.15 in D₂ O, each with integration values of 1H,were identified as vinyl protons. Also, in contrast to the methyldoublet at δ=0.9 ppm in compound 1360, the new steroid showed a singletmethyl at δ=1.8 ppm, characteristic of an allylic methyl. The chemicalshifts for the vinyl groups and for the allylic methyl compare favorablywith literature values. Otherwise, the ¹ H spectrum showed featurescharacteristic of compound 1360, including the presence of methylenesignals at δ=2.75 ppm, alpha to carbonyl at position 24. The polyamineregions exhibited splitting patterns like that of squalamine, confirmingthe spermidine adduct.

An accurate mass of 543.4823 was measured by FAB-MS (positive ion mode).The molecular formula C₃₄ H₆₁ N₃ O₂ has a calculated mass of 543.4842,in good agreement with the experimentally observed value. This molecularformula for compound 1361 is consistent with elimination of sulfate fromthe parent molecule, compound 1360. Moreover, the molecular formula forcompound 1361 has a double bond equivalent (DBE) of 5.5, in comparisonto 5.0 for compound 1360 and 4.0 for compound 1256; this DBE value isconsonant with the additional unsaturation in compound 1361.

Compound 1436

This compound and the steroids described below were purified bysubjecting fractions from preparative C18 RP-HPLC to shallower CH3CNgradient conditions on smaller C18 columns. Although strong cationexchange chromatography and silica gel (SG) flash chromatographyfollowed by RP-HPLC had been used in the purification of compounds 1360and 1361, these protocols were not used when the pH lability wasrecognized.

Although compound 1436 elutes from C18 RP-HPLC with retention time onlyslightly faster than squalamine, its R_(f) =0.47 on TLC hints of achemical structure with significantly greater polarity than squalamineunder alkaline conditions (CH₂ Cl₂ :CH₃ OH:NH₄ OH 6:3:1).

The ¹ H NMR spectrum in D₂ O (400 MHz) revealed the polyamine regionsdiffering from that of squalamine. Both the splitting pattern andintegration resembled spermine rather than spermidine, i.e.N,N'-bis-3-aminopropyl-1,4-butane-diamine rather thanN-(3-aminopropyl)-1,4-butanediamine. Otherwise, the ¹ H spectrumappeared identical to that of squalamine: one proton at δ=4.15, the 24sulfate position; one proton at δ=3.85, corresponding to H7 alcoholicring position; three overlapping doublets in 0.85-0.95 ppm correspondingto methyl 21 and methyls 26 and 27. The identity of spermine wassupported by performing COSY in D₂ O, comparing crosspeak patterns tothat of reference standards of spermine (C₁₀ H₂₆ N₄) and spermidine (C₇H₁₉ N₃) as well as to that of squalamine in D₂ O. Although COSY spectraof the aminosterols generally do not give a complete 2-dimensional mapof crosspeaks in D₂ O and therefore cannot be relied on for completenearest neighbor assignments, the polyamine region did produce acomplete set of off-diagonal crosspeaks, which served reliably as thesignature pattern for discerning between spermidine and spermine.

The ¹³ C spectrum of compound 1436 showed 3 additional signals in D₂ O,but otherwise the carbon skeleton of the steroid was the same as forsqualamine in D₂ O. DEPT-135 (distortionless enhanced polarizationtransfer) was conducted such that methyl and methine signals were phasedas positive signals, methylene groups as negative signals, andquaternary carbons gave zero intensity. DEPT-135 of the compounddemonstrated that these 3 additional signals were methylenes (negative).

The molecular formula of C₃₇ H₇₂ N₄ O₅ S has a calculated mass of684.53017, in good agreement with the experimentally observed accuratemass of 684.5216 measured by high resolution FAB-MS (positive ion mode).The additional mass of 58 daltons (684.5 versus 627.5 for squalamine)was consistent with the presence of an extra 3-aminopropyl groupattributed to spermine. Furthermore, the even number mass for the parention is consonant with the nitrogen rule, which predicts a compoundhaving an even number of nitrogens. FAB-MS also showed fragmentationinto species both 80 and 98 mass units less than the (M+H)+ parent at685 amu (atomic mass units). These fragments represent loss of sulfatefollowed by dehydration, paralleling the structural lability ofsqualamine under FAB-MS conditions.

Compound 1436 was also synthesized from compound 1256 (squalamine)according to the following reaction scheme: ##STR2##

Into a round-bottom flask was introduced 95 mg (0.106 mmol) ofsqualamine (TFA salt), which was dissolved in 800 μl of anhydrousmethanol. To the mixture was added 118 μl (0.848 mmol) of triethylamine, followed by 100 μl (0.106 mmol) of diluted acrylonitrile solution(70 μl acrylonitrile diluted to 1000 μl in methanol). After 6 hours, afurther 40 μl (0.042 mmol) of the dilute acrylonitrile solution wasadded. After 24 hours, TLC showed the presence of starting material anda product with R_(f) =0.7 (squalamine R_(f) =0.5). The reaction wasstopped, and the product isolated by flash chromatography (12:3:1 to6:3:1 CH₂ Cl₂ :MeOH:NH₄ OH).

The product, although homogenous by TLC, appeared to be a mixture by NMRspectroscopy. The product thus obtained was added to a hydrogenationflask along with 10 mg of Raney nickel, 7.3 mg of sodium hydroxide and 5ml of absolute ethanol, and hydrogenated at 40 psi for 17 hours. Twoproducts, now separable (flash chromatography, 6:3:1 CH₂ Cl₂ :MeOH:NH₄OH), were seen on TLC, wherein the lower spot co-spotted with thereference (naturally isolated compound 1436). This product was separatedby reverse-phase chromatography to yield 1.5 mg of pure material. Thiscompound had a positive mass (M+1) ion of 685, and its ¹ H and ¹³ C NMRspectra were identical to those of the naturally isolated material, thusconfirming its characterization and structure.

Compound 1437

This steroid, eluting immediately after compound 1360 in preparativeC18, exhibits R_(f) =0.96 on TLC, reflecting a more polar character thansqualamine itself. ¹ H NMR (400 MHz) in D₂ O appeared essentiallyidentical to squalamine for the methyl region, steroid nucleus andspermidine region, and 7H at the ring hydroxyl position. Conspicuouslyabsent from the ¹ H spectrum was the multiplet at δ=4.15 ppmcorresponding to one proton at the 24-sulfate position. Instead, a newsignal centered at δ=3.95 was observed with the characteristic gemalcohol coupling and integration for 2 protons.

When compared to squalamine, the ¹³ C spectrum of compound 1437 in D₂ Orevealed only two noticeable changes. One new signal appeared at δ=72ppm, which was subsequently identified as a --CH₂ OH group since itsDEPT-135 signal was negative. In squalamine, the sulfate position wasidentified at δ=86 ppm as a primary carbon (positive DEPT-135 signal).For compound 1437, however, this carbon resonance for the sulfateposition shifted to 76 ppm and gave no DEPT-135 signal, therebyidentifying it as a quaternary carbon. The aminosterol structureconsistent with these data has the carbon skeleton of ergostanol, withcarbon 24 bearing the sulfate and carbon 24' being the alcohol.

FAB-MS in positive ion mode indicated (M+H)+ at 658.6, fragmenting to578.6 due to loss of sulfate; negative ion mode analysis confirmed apseudomolecular parent ion (M-H)- at 656.4. An accurate mass of 578.5264was determined on the dessulfate fragment, on account of the lowintensity of the parent signal. The accurate mass of the parent ioncould then be calculated as 657.526 (by adding the mass of sulfate).Compound 1437 is thus 30 daltons greater than squalamine, which could beexplained by an additional --CH₂ OH moiety.

Steroids in Fraction I

Fraction I (FX1) is the earliest steroid fraction eluting frompreparative C18 RP-HPLC. TLC analysis typically showed a single majorspot with R_(f) =0.80-0.84 (with respect to squalamine, R_(f) =1.0) andprotein which stayed at the TLC origin. If the TLC plates were run withconcentrated samples (≧3 mg/ml), hints of additional spots, with R_(f)values either slightly greater than or less than the major component,were discernable.

When subjected to high resolution RP-HPLC using C18 columns with 60-100Å pore size and very shallow CH₃ CN gradients, Fraction I could beseparated into as many as 7 components, designated I-1, I-2, I-3, I-4,I-5, I-6, and 1-7. Steroids FX1A, FX1B, FX1C, FX1D are presented aspossible structures: ##STR3##

EXAMPLE 2

Synthesis of Aminosterols

In addition to the above compounds, which were isolated from sharkliver, synthetic aminosterol compounds have been developed. Variouspolyaminosterol compounds, including those specified in Examples A-G,are described in U.S. Ser. No. 08/416,883, which is the U.S. nationalphase of International Application No. PCT/US94/10265, filed Sep. 14,1994, the disclosure of which is herein incorporated by reference.Compounds exemplified therein include the following: ##STR4##

Additional aminosterol compounds have now been developed. Preferredcompounds of the invention include those exemplified below.

Example H Preparation of compound 353 and compound 354 ##STR5##

The above compounds were prepared by reductive coupling of5α-cholestan-3-one to spermine (4 equivalents) with sodiumcyanoborohydride in a manner analogous to the preparation of compound303. Purification was achieved on silica gel (gradient elution with9:3:1 to 3:3:1 chloroform:methanol:isopropylamine). Compound 353 (morepolar) and compound 354 (less polar) were converted to theirhydrochloride salts in the same manner as for compound 303. α-Aminocompound 354: ¹ H NMR (200 MHz, CD₃ OD) δ: 3.47 (m, 1H), 3.3-2.9 (m,12H), 2.3-1.0 (m, 39H), 1.0-0.8 (m, 12H), 0.70 (s, 3H); IR (KBr, cm⁻¹):3396, 2934, 1594, 1457, 1383; MS(+FAB): 573.6 (M+1); Anal. calcd. forC₃₇ H₇₂ N₄ -4HCl-H₂ O: C=60.31, H=10.67, N=7.60; Found: C=60.01,H=10.83, N=7.67. β-Amino compound 353: ¹ H NMR (200 MHz, CD₃ OD) δ:3.3-3.0 (m, 13H), 2.2-1.0 (m, 39H), 1.0-0.8 (m, 12H), 0.70 (s, 3H); IR(KBr, cm⁻¹): 2945, 1596, 1466, 1383; MS exact mass (+FAB) calcd.:573.5835; Found: 573.5801; Anal. calcd. for C₃₇ H₇₂ N₄ -4HCl-H₂ O:C=58.87, H=10.68, N=7.42; Found: C=58.49, H=10.94, N=7.94.

Compound 353 is a simple adduct of spermine and cholestanol,representing a very inexpensive compound. It can be synthesized likecompound 354 in the following straightforward manner: ##STR6##

Example I

Preparation of compound 458 and compound 459: ##STR7##

The above compounds were prepared from methyl 3-oxo-5α-cholanoate andspermine (1.35 equivalents) as in the synthesis of compound 353.Purification on silica gel (gradient elution with 6:3:1 to 3:5:2chloroform:methanol:isopropylamine) afforded the less polar α-aminocompound 458 and the more polar β-amino compound 459. These compoundswere converted to their hydrochloride salts as done as for compound 303.Compound 458: ¹ H NMR (400 MHz, CD₃ OD) δ: 3.64 (s, 3H), 3.45 (m, 1H),3.25-3.05 (m, 12H), 2.4-1.0 (m, 36H), 0.93 (d, J=6 Hz, 3H), 0.87 (s,3H), 0.70 (s, 3H); IR (KBr, cm⁻¹): 2943, 1741, 1458, 1169; MS(+FAB):575.6 (M+1); Anal. calcd. for C₃₅ H₆₆ N₄ O₂ -4HCl-1.2H₂ O: C=56.63,H=9.83, N=7.55; Found: C=56.58, H=9.46, N=7.29. Compound 459: ¹ H NMR(400 MHz, CD₃ OD) δ: 3.63 (s, 3H), 3.2-3.0 (m, 13H), 2.4-1.0 (m, 36H),0.92 (d, J=6 Hz, 3H), 0.86 (s, 3H), 0.69 (s, 3H); IR (KBr, cm⁻¹): 2942,1739, 1595, 1459, 1382, 1170; MS(+FAB): 575.6 (M+1); Anal. calcd. forC₃₅ H₆₆ N₄ O₂ -4HCl-1.4H₂ O: C=56.35, H=9.84, N=7.51; Found: C=56.35,H=9.26, N=7.67.

Example J

Preparation of compounds 380, 381, 382 and 394: ##STR8##

The steroid methyl 7α-hydroxy-3-oxo-5α-cholanoate was prepared accordingto the method of Iida et al., Chem. Pharm. Bull. 41(4), 1993, 763-765.This steroid was coupled to the polyamine compound 301 with sodiumcyanoborohydride, the BOC groups were removed with trifluoroacetic acid,and the ester was hydrolyzed as in the preparation of compound 319,except that lithium hydroxide was used as the base. Purification wasachieved on silica gel (15:4:1 to 10:4:1chloroform:methanol:isopropylamine). Compounds 381 and 382 were treatedwith 2M ammonia in methanol and evaporated (3×20 ml) to drive offisopropylamine. The hydrochloride salt was prepared as for compound 303.

Compound 380, C₃₂ H₅₉ N₃ O₃ : ¹ H NMR (200 MHz, CDCl₃) δ: 3.83 (br s,1H), 3.66 (s, 3H), 2.8-2.4 (m, 9H), 2.3-1.0 (m, 32H), 0.92 (d, J=6 Hz,3H), 0.78 (s, 3H), 0.65 (s, 3H); IR (KBr, cm⁻¹): 3278, 2928, 1736, 1447,1163; MS(+FAB): 534 (M+1).

Compound 381, C₃₁ H₅₇ N₃ O₃ -1.7 H₂ O; ¹ H NMR (200 MHz, CD₃ OD) δ: 3.80(br s, 1H), 3.0-2.5 (m, 9H), 2.2-1.1 (m, 32H), 0.94 (d, J=6 Hz, 3H),0.84 (s, 3H), 0.69 (s, 3H) ; IR (KBr, cm⁻¹): 3380, 2929, 1560, 1395;MS(+FAB) calcd.: 520.4478 (M+1); Found: 520.4506; Anal. calcd.: C=67.64,H=11.06, N=7.63; Found: C=67.64, H=10.24, N=7.83.

Compound 382, C₃₁ H₅₇ N₃ O₃ -2H₂ O: ¹ H NMR (200 MHz, CD₃ OD) δ: 3.80(br s, 1H), 3.15 (br s, 1H), 3.1-2.6 (m, 8H), 2.2-1.1 (m, 32H), 0.96 (d,J=6 Hz, 3H), 0.85 (s, 3H), 0.69 (s, 3H); IR (KBr, cm⁻¹): 3416, 2930,1560, 1395; MS(+FAB) calcd.: 520.4478 (M+1); Found: 520.4489; Anal.calcd: C=66.99, H=11.06, N=7.56; Found: C=66.93, H=10.16, N=7.28.

Compound 394, C₃₂ H₅₉ N₃ O₃ -3HCl-0.5H₂ O: ¹ H NMR (200 MHz, CD₃ OD) δ:3.83 (br s, 1H), 3.64 (s, 3H), 3.48 (br s, 1H), 3.3-2.9 (m, 8H), 2.4-1.1(m, 32H), 0.94 (d, J=6 Hz, 3H), 0.87 (s, 3H), 0.70 (s, 3H); MS(+FAB):535 (M+1); Anal. calcd.: C=58.93, H=9.74, N=6.44; Found: C=58.71,H=10.13, N=6.39.

Example K

Preparation of compounds 395, 396 and 397: ##STR9##

Methyl 7α-hydroxy-3-oxo-5α-cholanoate was coupled to spermine (2equivalents) with sodium cyanoborohydride, and the ester was hydrolyzedas in the preparation of compound 319, except that lithium hydroxide wasused as the base. Purification of compounds 395 and 396 was achieved onsilica gel (15:5:1 to 5:5:1 chloroform:methanol:isopropylamine).Purification of compound 397 was achieved on silica gel (2:6:1benzene:methanol:isopropylamine), followed by treatment with 2M ammoniain methanol (3×20 ml) to drive off isopropylamine. The hydrochloridesalts of compounds 395 and 396 were prepared in the same manner as forcompound 303.

Compound 395, C₃₅ H₆₆ N₄ O₃ -4HCl-2H₂ O: ¹ H NMR (200 MHz, CD₃ OD) δ:3.80 (br s, 1H), 3.64 (s, 3H), 3.3-3.0 (m, 13H), 2.4-1.0 (m, 34H), 0.94(d, J=6 Hz, 3H), 0.87 (s, 3H), 0.70 (s, 3H); Anal. calcd.: C=54.40,H=9.65, N=7.25; Found: C=54.16, H=9.31, N=7.12.

Compound 396, C₃₅ H₆₆ N₄ O₃ -4HCl-0.5H₂ O: MS(+FAB): 592 (M+1); Anal.calcd.: C=56.37, H=9.60, N=7.51; Found: C=56.43, H=9.83, N=7.27.

Compound 397, C₃₄ H₆₄ N₄ O₃ : ¹ H NMR (200 MHz, CD₃ OD) δ: 3.78 (br s,1H), 2.9-2.5 (m, 13H), 2.2-1.1 (m, 34H), 0.95 (d, J=6 Hz, 3H), 0.87 (s,3H), 0.70 (s, 3H); MS(+FAB): 577.3 (M+1).

Example L

Preparation of compound 393: ##STR10##

Compound 304 (210 mg, 0.41 mmol) was dissolved in methanol (10 ml) undernitrogen, and treated with o-methylisourea hydrochloride (50 mg, 0.45mmol) and 1N sodium hydroxide solution (0.45 ml, 0.45 mmol). After 23hours, additional o-methylisourea was added (102 mg, 0.92 mmol), and thereaction was continued for 7 hours, quenched with 1N hydrochloric acidsolution (pH<7), and evaporated. The residue was partitioned between 1Nsodium hydroxide solution (50 ml) and chloroform (100 ml). After washingwith additional chloroform (50 ml), the combined organic layers weredried (Na₂ SO₄) and concentrated. Purification by flash chromatographyon silica gel (2-cm diameter, gradient elution with 5 to 15% methanol inmethylene chloride) afforded a white solid (32 mg). This material wasdissolved in chloroform (3 ml), cooled in an ice bath, treated with 1Mhydrogen chloride in ether (1 ml), and concentrated in vacuo to affordcompound 393 (37 mg, 14% yield). C₃₅ H₆₄ N₄ -2HCl-2H₂ O: ¹ H NMR (200MHz, CD₃ OD) δ: 3.5-3.3 (m, 5H), 3.2-3.0 (m, 4H), 2.2-1.0 (m, 37H),0.95-0.86 (m, 9H), 0.70 (s, 3H); IR (KBr, cm⁻¹): 3306, 3153, 2934, 1654,1586, 1445, 1383; MS(+FAB): 541.4 (M+1); Anal. calcd.: C=64.69, H=10.86,N=8.62, Found: C=65.06, H=10.98, N=8.83.

Example M

Preparation of compounds 370 and 371: ##STR11##

Preparation of compound 1010: ##STR12##

To a suspension of pyridinium chlorochromate (6.85 g, 31.8 mmol) indichloromethane (130 ml) was added a solution of compound 1006 (5.96 g,13.3 mmol) in dichloromethane (70 ml). After stirring for 3 hours atroom temperature, the reaction mixture was diluted with ether (100 ml),filtered, and washed with ether. The organic layer was washed with 5%sodium hydroxide solution, 5% hydrochloric acid solution, saturatedsodium bicarbonate and brine. The dried ethereal layer was evaporatedand purified by flash chromatography (6 cm, gradient elution with 0-20%ethyl acetate in hexane) to yield pure compound 1010 (5.25 g, 77%yield). ¹ H NMR (200 MHz, CDCl₃) δ: 4.92 (m, 1H), 2.5-1.0 (m, 29H), 2.06(s, 3H), 1.03 (s, 3H), 0.91 (d, J=6.5 Hz, 3H), 0.87 (d, J=6.5 Hz, 6H),0.67 (s, 3H); IR KBr, cm⁻¹); 2949, 1736, 1468, 1372, 1244, 1023;MS(ES+): 467.8 (M+Na).

Preparation of compounds 370 and 371: The steroid 1010 was coupled topolyamine 301 with sodium cyanoborohydride, the BOC groups were removedwith trifluoroacetic acid, and the acetate was hydrolyzed as in thepreparation of compound 319, except that sodium hydroxide was used asthe base. Purification was achieved on silica gel (2:6:1benzene:methanol:isopropylamine). Compound 370 (¹ H NMR (200 MHz, CD₃OD) δ: 3.80 (m, 1H), 2.97 (m, 1H), 2.9-2.6 (m, 8H), 2.1-1.0 (m, 35H),0.94 (d, J=6.5 Hz, 3H), 0.88 (d, J=6.5 Hz, 6H), 0.87 (s, 3H), 0.70 (s,3H)) and compound 371 (¹ H NMR (200 MHz, CD₃ OD) δ: 3.77 (m, 1H),2.8-2.5 (m, 9H), 2.1-1.0 (m, 35H), 0.93 (d, J=6.5 Hz, 3H), 0.88 (d,J=6.5 Hz, 6H), 0.83 (s, 3H), 0.69 (s, 3H)) were converted to theirhydrochloride salts as in the preparation of compound 303. Compound 370:IR (KBr, cm⁻¹): 3415, 2948, 1595, 1467, 1382, 1031; MS(+FAB): 532.4(M+1); Anal. calcd. for C₃₄ H₆₅ N₃ O-0.3HCl·2H₂ O: C=60.29, H=10.71,N=6.20; Found: C=60.01, H=11.09, N=6.3. Compound 371: IR (KBr, cm⁻¹):3414, 2953, 1596, 1468, 1381, 1033; MS(+FAB): 532.4 (M+1); Anal. calcd.for C₃₄ H₆₅ N₃ 0.3HCl·2H₂ O: C=60.29, H=10.71, N=6.20; Found: C=60.49,H=11.00, N=6.47.

Example N

Preparation of compound 470: ##STR13##

Preparation of precursors: ##STR14##

Preparation of compounds 1011 and 1012: A solution of methyl3-oxo-5α-cholanoate (compound 310, 2.00 g, 5.15 mmol), p-toluenesulfonicacid (250 mg), and ethylene glycol (25 ml) in benzene (160 ml) washeated to reflux with the removal of water for 6 hours. After cooling toroom temperature, saturated sodium bicarbonate (30 ml) was added, andthe aqueous phase was extracted with benzene and ethyl acetate. Theorganic layers were washed with water and brine, dried over sodiumsulfate, and evaporated to yield compound 1011, which was used for thenext step without purification.

A solution of 1M lithium aluminum hydride (25 ml, 25 mmol) in etherunder nitrogen was treated with a solution of compound 1011 in anhydrousether (80 ml) and heated to reflux for 5 hours. After stirringovernight, the reaction mixture was quenched at 0° C. with water and 2Nsodium hydroxide solution. The aqueous layer was extracted with ether,followed by washing with brine, drying over magnesium sulfate, andevaporating to afford compound 1012 (1.80 g, 86% yield). ¹ H NMR (400MHz, CDCl₃) δ: 3.94 (s, 4H), 3.62 (m, 2H), 2.0-1.0 (m, 28H), 0.92 (d,J=6 Hz, 3H), 0.81 (s, 3H), 0.66 (s, 3H).

Preparation of compounds 1013 and 1014: A solution of compound 1012(3.63 g, 8.97 mmol) in anhydrous pyridine (16 ml) was treated withp-toluenesulfonyl chloride (2.3 g, 12.1 mmol) at room temperature, andleft overnight. Ice water was added, and the reaction mixture was leftfor 30 minutes with stirring. Then 6N hydrochloric acid was added (70ml), and the aqueous layer was extracted with dichloromethane and ether.The organic layers were washed with 2N hydrogen chloride, saturatedsodium bicarbonate and brine, dried, and evacuated to yield crudecompound 1013. Compound 1013 was dissolved in dimethylsulfoxide (40 ml)and treated with sodium cyanide (1.4 g, 28 mmol) at 90° C. for 2.5 hoursunder nitrogen. After cooling, the reaction mixture was treated with icewater and extracted into ether and dichloromethane. The organic layerswere washed with brine, dried over sodium sulfate, and purified bychromatography (4-cm diameter, gradient elution with 0-25% ethyl acetatein hexane) to yield pure compound 1014. ¹ H NMR (400 MHz, CDCl₃) δ: 3.94(s, 4H), 2.32 (m, 2H), 2.0-1.0 (m, 28H), 0.93 (d, J=6 Hz, 3H), 0.81 (s,3H), 0.66 (s, 3H); IR (KBr, cm⁻¹): 2930, 2247, 1445, 1381, 1357, 1133,1091, 928, 899; MS(+FAB): 414.4 (M+1).

Preparation of compound 1015: A solution of compound 1014 (480 mg, 1.16mmol) in acetic acid (35 ml) and concentrated hydrochloric acid (25 ml)was refluxed for 25 hours. After evaporating the solvent, the residuewas partitioned between water and ethyl acetate. After drying andevaporating, the crude carboxylic acid was dissolved in methanol (25ml), treated with concentrated hydrochloric acid (1 ml), and brought toreflux for 20 minutes. After evaporation of solvent, the product wasdissolved in ethyl acetate and water and extracted again with ethylacetate. The organic layers were washed with brine, dried over sodiumsulfate, and purified by flash chromatography (2-cm diameter, gradientelution with 0-25% ethyl acetate in hexane) to afford pure compound 1015(298 mg, 64% yield), m.p. 147°-148° C. ¹ H NMR (400 MHz, CDCl₃) δ: 3.67(s, 3H), 2.4-1.0 (m, 30H), 1.01 (s, 3H), 0.93 (d, J=6 Hz, 3H), 0.68 (s,3H); ¹³ C NMR (400 MHz, CDCl₃) δ: 212.3, 174.5, 56.5, 56.1, 54.0, 51.6,46.9, 44.9, 42.8, 40.1, 38.8, 38.4, 35.8, 35.7, 35.6, 34.7, 31.9, 29.2,28.4, 24.4, 21.7, 21.6, 18.8, 12.2, 11.7; MS(+FAB): 403.3 (M+1); Anal.calcd. for C₂₆ H₄₂ O₃ : C=77.56, H=10.51; Found: C=77.49, H=10.52.

Preparation of compound 470: Steroid 1015 was coupled to polyamine 301with sodium cyanoborohydride, the BOC groups were removed withtrifluoroacetic acid, and the ester was hydrolyzed as in the preparationof compound 319, except that lithium hydroxide was used as the base.Purification was achieved on silica gel (gradient elution with 14:4:1 to4:4:1 chloroform:methanol:isopropylamine). After evaporation frommethanol:chloroform (3x), the compound was treated with 2M ammonia inmethanol and evaporated (3×20 ml) to drive off isopropylamine. ¹ H NMR(400 MHz, CD₃ OD) δ: 2.8-2.6 (m, 9H), 2.2-1.0 (m, 36H), 0.92 (d, J=6 Hz,3H), 0.80 (s, 3H), 0.66 (s, 3H); MS(+FAB): 518.4 (M+1); Anal. calcd.:C=71.73, H=11.47, N=7.84; Found: C=2.03, H=11.06, N=7.53.

Example Q

Preparation of compounds 431, 432, 433, 465, 466, 467, and 469.##STR15##

Preparation of precursors: ##STR16##

Preparation of compound 1016: The methyl ester of hyodeoxycholic acidwas prepared by acid-catalyzed esterification of hyodeoxycholic acid inmethanol. To a magnetically stirred 500-ml round-bottom flask containingabsolute methanol (200 ml) was added hyodeoxycholic acid (10 g, 25.5mmol) and concentrated sulfuric acid (5 ml) dropwise. The reaction wasstirred overnight and then treated with dichloromethane (250 ml),followed by washing with sodium bicarbonate solution (2×100 ml) andbrine (100 ml). The organic layer was then dried over anhydrous sodiumsulfate, filtered, and dried under vacuum to yield compound 1016 (10.1g, 97% yield) (see Organic Preparations and Procedures Int. 19(2-3),1987, 197-208).

Preparation of compound 1017: The 3,6-dioxo sterol was prepared byoxidation of methyl hyodeoxycholic acid with pyridinium chlorochromate.Compound 1016 (10.1 g, 25 mmol) was dissolved in dichloromethane (200ml). To a magnetically stirred flask in an ice water bath was addedpyridinium chlorochromate (33 g, 150 mmol). The reaction was allowed towarm to room temperature and to proceed for 8 hours, until the productwas the only visible TLC spot. A major portion of the dichloromethanewas removed under vacuum, and ethyl acetate (250 ml) was then added tothe flask. The chromium crust in the bottom of the flask was broken upwith a spatula, and the contents of the flask were filtered through aCelite column. The elutant from the column was then reduced in volumeunder vacuum and filtered through a florisil column (elution with ethylacetate). The elutant was again reduced in volume to approximately 200ml, and diethyl ether (100 ml) was added, followed by washing withsodium bicarbonate solution (2×250 ml) and then brine (250 ml). Theorganic layer was dried over anhydrous sodium sulfate, filtered, anddried under vacuum. The total yield of methyl3,6-dioxo-5β-cholan-24-oate 1017 without recrystallization was 9.6 g (24mmol, 96%) (see Organic Preparations and Procedures Int. 19(2-3), 1987,197-208). The product can be recrystallized from a number of solvents(absolute methanol, ethyl acetate in hexanes, or diethyl ether inhexanes) if any chromium remains.

Preparation of compound 1018: The 3,6-dioxo-5α sterol was prepared byacid-catalyzed isomerization of the 5β sterol. To methanol (250 ml) wasadded the 3,6-dioxo-5β sterol 1017 (9.6 g, 24 mmol) and tetrahydrofuran(25 ml) to dissolve the sterol completely. Concentrated hydrochloricacid (12.5 ml) was added, and the reaction was allowed to proceedovernight. The solvent was then removed under vacuum to yield 9.6 g(100% yield) of methyl 3,6-dioxo-5α-cholan-24-oate 1018 (see OrganicPreparations and Procedures Int. 19(2-3), 1987, 197-208; authors usedbase-catalyzed isomerization using sodium methoxide rather than HCl).

Preparation of compound 1019: The mono-protection of methyl3,6-dioxo-5α-cholan-24-oate 1018 may be accomplished using a variety oftechniques. One technique involved refluxing compound 1018 (9.6 g, 23.8mmol) in toluene (250 ml) with ethylene glycol (1.77 g, 28.5 mmol) inthe presence of catalytic p-toluenesulfonic acid. A Dean Stark trap wasused for removing the toluene/water azeotrope. The reaction was judgedto be complete by TLC after approximately 20 minutes. The reaction wasworked up by pouring the toluene over sodium bicarbonate solution (500ml) and ice slurry. The organic layer was washed with additional sodiumbicarbonate (200 ml) and brine (200 ml), dried over anhydrous sodiumsulfate, filtered, and dried under vacuum. The crude product waschromatographed on silica gel (4 cm×25 cm, elution with 33% ethylacetate in hexanes). Methyl 3-dioxolane-6-oxo-5α-cholan-24-oate 1019(8.9 g, 81) was the second band off the column; the only other productpresent was the less polar di-dioxolane. Subsequent techniques yieldedbetter results by substituting benzene for toluene and following thereaction by TLC, which apparently allows for greater selectivity. Thereaction can be stopped before significant di-protection occurs in thelower boiling solvent. Compound 1019: m.p. 124°-126° C.; ¹ H NMR (200MHz, CDCl₃) δ: 4.04-3.93 (m, 4H), 3.68 (s, 3H), 0.95 (d, J=6 Hz, 3H),0.78 (s, 3H), 0.69 (s, 3H); IR (KBr, cm⁻¹): 2945, 1742, 1709, 1439,1381, 1313, 1162, 1090; MS(FD): 446 (M⁺), 388.

Preparation of compound 1020: The 6β-hydroxy sterol was prepared in goodyield from the mono-protected diketone by reduction with sodiumborohydride. The 3-dioxolane-6-oxo sterol 1019 (5 g, 11 mmol) wasdissolved in tetrahydrofuran (10 ml) and added to absolute methanol (200ml) and sodium borohydride (2.5 g, 66 mmol). The sodium borohydride wasdissolved and stirred for approximately 20-30 minutes before theaddition of the sterol. After stirring overnight, the reaction mixturewas treated with chloroform (500 ml), and washed with distilled water(2×200 ml) and then brine (100 ml). The organic layer was then driedover sodium sulfate, filtered, concentrated under vacuum, and purifiedby flash chromatography on silica gel (4 cm×25 cm, elution with 2:1:1hexanes:ethyl acetate:methylene chloride) to yield methyl3-dioxolane-6β-hydroxy-5α-cholan-24-oate 1020 (4.35 g, 87% yield).Alternatively, the crude product can be recrystallized from benzene inhexanes, ethyl acetate in hexanes, or chloroform in hexanes (2x) toyield a product of high purity without need for column chromatography.Compound 1020: m.p. 164° C.; ¹ H NMR (200 MHz, CDCl₃) δ: 4.04-3.93 (m,4H), 3.77 (br s, 1H), 3.66 (s, 3H), 1.03 (s, 3H), 0.92 (d, J=6 Hz, 3H),0.69 (s, 3H); IR (KBr, cm⁻¹): 3533, 2937, 1726, 1438, 1379, 1255, 1191,1096; X-ray diffraction revealed the expected structure.

Preparation of compound 1021: The 3-dioxolane was deprotected usingacidic acetone solution. The 3-dioxolane-6β-hydroxy-sterol 1020 (4.0 g,8.9 mmol) was dissolved in acetone (200 ml) and treated withconcentrated hydrochloric acid solution (10 ml). After approximately 1hour, the reaction mixture was poured into a sodium bicarbonatesolution. The solution was extracted with dichloromethane (3×200 ml),washed with distilled water (100 ml) and then brine (100 ml), dried overanhydrous sodium sulfate, filtered, and evaporated under vacuum to yieldmethyl 3-oxo-6β-hydroxy-5α-cholan-24-oate 1021 (3.45 g, 100% yield): ¹ HNMR (200 MHz, CDCl₃) δ: 3.8 (br m, 1H), 3.69 (s, 3H), 1.24 (s, 3H), 0.95(d, J=6 Hz, 3H), 0.74 (s, 3H); IR (KBr, cm⁻¹): 3447, 2954, 1742, 1707,1431.

Preparation of compounds 431 and 432: The ethylene-diamine compoundswere prepared as follows. A magnetically stirred solution of 50:50methanol:tetrahydrofuran (100 ml) and ethylenediamine (2 ml) was treatedwith acetic acid to lower the pH to approximately 6. The 3-oxo sterol1021 (1.5 g, 3.7 mmol) was added, and the mixture was stirred for 15minutes. Sodium cyanoborohydride (1 g, 16 mmol) was dissolved in 10 mlmethanol and added to the reaction vessel, and the pH was again adjustedto 6 by the addition of acetic acid. The reaction was stirred for 1hour, and the contents of the flask were poured into a pH 10.5carbonate-buffer ice slurry (250 ml). The solution was extracted withchloroform (5×150 ml). The organic layers were combined, dried overanhydrous sodium sulfate, filtered, dried under vacuum, and purified byflash chromatography on silica gel (4 cm×25 cm, elution with 8:2:1chloroform:methanol:isopropylamine) to afford the less polar α-isomer431 (260 mg, 15% yield) and the more polar β-isomer 432 (840 mg, 49%yield). Compound 431: ¹ H NMR (400 MHz, CD₃ OD) δ: 3.74 (m, 1H), 3.65(s, 3H), 3.53 (m, 1H), 1.06 (s, 3H), 0.94 (d, J=6 Hz, 3H), 0.74 (s, 3H);IR (KBr, cm⁻¹): 3426, 2943, 1740, 1590, 1438, 1379, 1258, 1168, 1027;MS(+FAB): 449.5 (M+1); Anal. calcd. for C₂₇ H₄₈ N₂ O₃ -2HCl-0.7H₂ O:C=60.70, H=9.70, N=5.24; Found: C=60.97, H=9.68, N=5.34. Compound 432: ¹H NMR (400 MHz, CD₃ OD) δ: 3.75 (m, 1H), 3.64 (s, 3H), 1.02 (s, 3H),0.94 (d, J=6 Hz, 3H), 0.73 (s, 3H); IR (KBr, cm⁻¹): 3560, 3366, 3257,2936, 1726, 1648, 1605, 1438, 1376, 1166, 1047; MS(+FAB): 449.5 (M+1);Anal. calcd. for C₂₇ H₄₈ N₂ O₃ -0.4H₂ O: C=71.13, H=10.79, N=6.14;Found: C=71.15, H=10.71, N=6.28.

Preparation of compounds 465 and 466: To a magnetically stirred flaskcontaining anhydrous methanol (100 ml) was added compound 1021 (1.5 g,3.7 mmol), spermine (2 g, 9.9 mmol), powdered 3 Å sieves (2 g), andacetic acid until the pH was 6. The flask was sealed, the contentsstirred overnight, and then sodium cyanoborohydride (1 g, 16 mmol) inmethanol (10 ml) was added. The pH was again adjusted with acetic acid,and the reaction mixture was stirred for 8 hours. The workup was similarto the workup for the ethylenediamine compounds. The crude product waspurified by flash chromatography (5 cm×25 cm, elution with 4:5:1chloroform:methanol:isopropylamine), affording less polar α-amino isomer465 and more polar β-amino isomer 466. The total yield of amino sterolwas 1.3 g (58% yield). Compound 465: ¹ H NMR (400 MHz, CD₃ OD) δ: 3.75(m, 1H), 3.65 (s, 3H), 3.54 (m, 1H), 1.06 (s, 3H), 0.95 (d, J=6 Hz, 3H),0.74 (s, 3H); IR (KBr, cm⁻¹): 3406, 2944, 1740, 1596, 1466, 1168, 1049,1027; MS(+FAB): 591.4 (M+1); Anal. calcd. for C₃₅ H₆₆ N₄ O₃ -4HCl-1.2H₂O: C=55.43, H=9.62, N=7.39; Found: C=55.70, H=9.15, N=7.12. Compound466: ¹ H NMR (400 MHz, CD₃ OD) δ: 3.79 (m, 1H), 3.65 (s, 3H), 1.06 (s,3H), 0.95 (d, J=6 Hz, 3H), 0.74 (s, 3H); IR (KBr, cm⁻¹): 3406, 2944,1740, 1595, 1459, 1381, 1167, 1051, 1026; MS(+FAB): 591.4 (M+1); Anal.calcd. for C₃₅ H₆₆ N₄ O₃ -4HCl-1.2H₂ O: C=55.43, H=9.62, N=7.39; Found:C=55.48, H=9.03, N=7.33.

Preparation of compound 469: This compound was prepared in a manneranalogous to that used for compound 466, but using polyamine 1023:##STR17## The polyamine was prepared from piperazine by double additionof acrylonitrile to yield compound 1022, which was reduced by Raneynickel catalyzed hydrogenation. β-amino isomer 469: ¹ H NMR (400 MHz,CD₃ OD) δ: 3.78 (m, 1H), 3.64 (s, 3H), 3.5-3.3 (m, 8H), 3.2-3.0 (m, 9H),2.4-1.0 (m, 30H), 1.03 (s, 3H), 0.92 (d, J=6.5 Hz, 3H), 0.71 (s, 3H); IR(KBr, cm⁻¹): 3406, 2943, 1736, 1594, 1443, 1165; MS(+FAB): 589.4 (M+1);Anal. calcd. for C₃₅ H₆₄ N₄ O₃ -4HCl-3H₂ O: C=53.29, H=9.46, N=7.10;Found: C=53.06, H=8.90, N=8.43.

Preparation of compounds 433 and 467: An amount of aminosterol methylester (1 mmol) as the free base was weighed into a 25-ml round bottomflask. The aminosterol was dissolved in a minimal amount oftetrahydrofuran (2 ml), treated with 1N potassium hydroxide solution (10ml), and magnetically stirred for 1 hour. The solution was thenneutralized with 1N HCl, and the solvent was removed under vacuum. Theresidue was redissolved in a minimal amount of deionized water andapplied to an octadecyl-functionalized silica gel column (Aldrich, 2×10cm, gradient elution of acetonitrile in 2% trifluoroacetic acid inwater). The fractions containing aminosterol were pooled, and thesolvent was removed under vacuum. The aminosterol was redissolved in0.1N HCl, and the solvent was removed under vacuum (2x) to insure theremoval of trifluoroacetate. Benzene was added to the resultinghydrochloride salts, followed by evaporation overnight to remove as muchwater as possible.

Ethylenediamine β-amino isomer 433 was not treated with HCl, butisolated as the trifluoroacetate salt. Compound 433: ¹ H NMR (400 MHz,CD₃ OD) δ: 3.78 (m, 1H), 1.06 (s, 3H), 0.95 (d, J=6.5 Hz, 3H), 0.74 (s,3H); IR (KBr, cm ): 3533, 3488, 2941, 1716, 1679, 1615, 1489, 1431,1191; MS(+FAB): 435.5 (M+1), 531.5 (likely a trace of thetrifluoroacetamide); Anal. calcd. for C₂₆ H₄₆ N₂ O₃ -2TFA-0.7H₂ O:C=53.36, H=7.37, N=4.15; Found: C=54.36, H=7.45, N=4.40.

Spermine β-amino isomer 467: ¹ H NMR (400 MHz, CD₃ OD) δ: 3.80 (m, 1H),1.05 (s, 3H), 0.95 (d, J 6.5 Hz, 3H), 0.73 (s, 3H); IR (KBr, cm⁻¹):3406, 2944, 1718, 1637, 1458; MS(+FAB): 577.4 (M+1); Anal. calcd. forC₃₄ H₆₄ N₄ O₃ -4HCl-4H₂ O: C=51.38, H=9.64, N=7.05; Found: C=51.40,H=8.77, N=7.01.

Example P

Preparation of bile acid methyl esters 409, 410, 411, 355, 356, 416,448, 414, 415, 412, 413, 417 and 449: ##STR18##

Preparation of precursors: The methyl esters of chenodeoxycholic acidand deoxycholic acid, which are structurally depicted below, wereprepared by the same procedure as used to esterify hyodeoxycholic acidto compound 1016. ##STR19##

Silver carbonate oxidations of bile acid esters to prepare 3-ketosteroids: Both chenodeoxycholic and deoxycholic acid derivatives wereprepared by reductive aminations of the 3-oxo sterols with theappropriate amines. The 3-oxo sterols were prepared by similarprocedures. Silver carbonate on Celite was prepared by dissolving 4equivalents of silver nitrate in deionized water and adding sufficientCelite to result in 50% silver carbonate on Celite. To the magneticallystirred solution was added 2.2 equivalents of sodium carbonate dissolvedin deionized water, with continued vigorous stirring. The resultingsilver carbonate precipitated on Celite was filtered through aglass-fritted funnel, washed with tetrahydrofuran, and allowed to dry ina vacuum desiccator. The methyl ester of the bile acid to be oxidizedwas dissolved in toluene, treated with 2 equivalents of silver carbonateon Celite, and heated to reflux using a Dean Stark apparatus forazeotropic removal of water. The oxidation was complete in less than 6hours for both sterols. The only product in both cases was the desired3-oxo sterol. The solution was filtered and the solvent removed undervacuum. The product in both cases recrystallized readily from ethylacetate in hexanes to give the 3-oxo sterol in excellent yield (>89% inboth cases).

Preparation of compounds 409 and 410: The 3-oxo sterol methyl ester ofchenodeoxycholic acid (1.5 g, 3.7 mmol) was dissolved in methanol, towhich a ten-fold excess of ethylenediamine (2.5 ml) was added. The pHwas lowered with acetic acid to approximately 6, NaBH₃ CN (1 g, 15.9mmol) dissolved in methanol was added, and the pH was again adjustedwith acetic acid. The solution was stirred for 1 hour, and then workedup and purified in the same manner as compound 431. The total yield ofaminosterol was 58%, with an approximate ratio of α-amino isomer to theless polar β-amino isomer of 7:3. β-Amino isomer 409: ¹ H NMR (400 MHz,CD₃ OD) δ: 3.81 (m, 1H), 3.68 (s, 3H), 3.42 (m, 1H), 1.04 (s, 3H), 0.95(d, J=6.5 Hz, 3H), 0.72 (s, 3H); IR (KBr, cm⁻¹): 3428, 2940, 2055, 1740,1591, 1440, 1377, 1169, 1077, 984; MS(+FAB): 449.3 (M+1); Anal. calcd.for C₂₇ H₄₈ N₂ O₃ -2HCl-1.2H₂ O: C=59.70, H=9.72, N=5.16; Found:C=59.59, H=9.49, N=5.15. β-Amino isomer 410: ¹ H NMR (400 MHz, CD₃ OD)δ: 3.82 (m, 1H), 3.65 (s, 3H), 3.05 (br m, 1H), 1.00 (s, 3H), 0.94 (d,J=6.5 Hz, 3H), 0.72 (s, 3H); IR (KBr, cm⁻¹): 3522, 2944, 2017, 1718,1619, 1448, 1377, 1314, 1282, 1260, 1163, 1018; MS(+FAB): 449.3 (M+1);Anal. calcd. for C₂₇ H₄₈ N₂ O₃ -2HCl-3.7H₂ O: C=55.13, H-9.84, N=4.76;Found: C=55.03, H=9.32, N=4.78.

Preparation of compound 411: This spermine compound was prepared by thesame procedure as the ethylenediamine compounds, except for thefollowing modification. One gram of the 3-oxo sterol methyl ester ofchenodeoxycholic acid and 1 g of spermine (approx. 2 equiv.) were used,and the chromatography required a more polar solvent system (i.e., 5:4:1CHCl₃ :methanol:isopropylamine). The total yield of aminosterol was 48%.The ratio of a-amino isomer to β-amino isomer 411 was not determined dueto incomplete separation. Compound 411: ¹ H NMR (400 MHz, CD₃ OD) δ:3.83 (m, 1H), 3.65 (s, 3H), 3.42 (m, 1H), 1.04 (s, 3H), 0.95 (d, J=6.5Hz, 3H), 0.70 (s, 3H); IR (KBr, cm⁻¹): 3404, 2946, 2059, 1739, 1595,1458, 1378, 1168, 1073, 1012, 985, 759; MS(+FAB): 591.4 (M+1); Anal.calcd. for C₃₆ H₆₆ N₄ O₃ -4HCl-4H₂ O: C=51.97, H=9.72, N=6.93; Found:C=51.65, H=8.53, N=6.77.

Preparation of compounds 355 and 356: The 3-oxo sterol methyl ester ofchenodeoxycholic acid was coupled to polyamine 301 with sodiumcyanoborohydride, the BOC groups were removed with trifluoroacetic acid,and the ester was hydrolyzed as in the preparation of compound 319.Purification was achieved on silica gel (15:4:1 to 10:4:1chloroform:methanol:isopropylamine). Less polar β-amino isomer 355, C₃₂H₅₉ N₃ O₃ : ¹ H NMR (400 MHz, CDCl₃) δ: 3.87 (m, 1H), 3.68 (s, 3H), 3.15(m, 1H), 3.0-2.7 (m, 8H), 2.4-1.0 (m, 32H), 0.99 (s, 3H), 0.91 (d, J=6Hz, 3H), 0.66 (s, 3H); MS(DCI): 534 (M+1). More polar α-amino isomer356, C₃₂ H₅₉ N₃ O₃ -3HCl: ¹ H NMR (400 MHz, CD₃ OD) δ: 3.82 (m, 1H),3.25-2.95 (m, 9H), 2.5-1.0 (m, 32H), 0.97 (s, 3H), 0.94 (d, J=6 Hz, 3H),0.69 (s, 3H); MS (DCI) 534 (M+1).

Preparation of compound 1416: The procedures used for the preparation ofdeoxycholic acid derivatives were the same as those used in thepreparation of the chenodeoxycholic acid derivatives. For theethylenediamine compound, the total yield of aminosterol was 62%, withthe ratio of α-amino isomer 416 to β-amino isomer being 4:1. Compound416: ¹ H NMR (400 MHz, CD₃ OD) δ: 3.97 (m, 1H), 3.68 (s, 3H), 3.22 (brm, 1H), 1.02 (d, J=6.5 Hz, 3H), 1.01 (s, 3H), 0.73 (s, 3H); IR (KBr,cm⁻¹): 3418, 2940, 1739, 1616, 1456, 1379, 1253, 1169, 1036; MS(+FAB):449.4 (M+1); C₂₇ H₄₈ N₂ O₃ -2HCl-0.5H₂ O: C=61.12, H=9.69, N=5.28;Found: C=61.20, H=9.50, N=5.07.

Preparation of compound 448: For the spermine derivatives of deoxycholicacid, the total yield of aminosterol was 46% (difficulty in the workupwas likely responsible for the lower yield). The ratio of α-amino isomer448 to β-amino isomer was not determined due to incomplete separation.Compound 448: ¹ H NMR (400 MHz, CD₃ OD) δ: 3.98 (m, 1H), 3.67 (s, 3H),1.01 (d, J=6 Hz, 3H), 1.01 (s, 3H), 0.73 (s, 3H); IR (KBr, cm⁻¹): 2944,1738, 1594, 1451, 1378, 1169, 1038, 758; MS(+FAB): 591.5 (M+1); Anal.calcd. for C₃₅ H₆₆ N₄ O₃ -4HCl-2.3H₂ O: C=54.02, H=9.66, N=7.20; Found:C=54.00, H=8.64, N=7.22.

Preparation of compounds 414 and 415: The free acids were prepared fromthe methyl esters as in the preparation of 6β-hydroxy 433. α-Aminoisomer 414: ¹ H NMR (400 MHz, CD₃ OD) δ: 3.83 (m, 1H), 3.06 (br m, 1H),1.04 (s, 3H), 0.96 (d, J=6 Hz, 3H), 0.73 (s, 3H); IR (KBr, cm⁻¹): 2940,2053, 1709, 1452, 1378, 1167, 1076, 1007, 975; MS(+FAB): 435.5 (M+1);Anal. calcd. for C₂₆ H₄₆ N₂ O₃ -2HCl-1.5H₂ O: C=58.41, H=9.62, N=5.24;Found: C=58.24, H=9.40, N=5.47. β-Amino isomer 415: ¹ H NMR (400 MHz,CD₃ OD) δ: 3.83 (m, 1H), 3.47 (m, 1H), 1.06 (s, 3H), 0.95 (d, J=6 Hz,3H), 0.73 (s, 3H); IR (KBr, cm⁻¹): 3488, 2935, 2054, 1709, 1593, 1499,1450, 1246, 1168, 1077, 1022, 984; MS(+FAB): 435.5 (M+1); Anal. calcd.for C₂₆ H₄₆ N₂ O₃ -2HCl-1.5H₂ O: C=58.41, H=9.62, N=5.24; Found:C=58.59, H=9.35, N=5.43.

Preparation of compounds 412, 413, 417 and 449: These compounds wereproduced using procedures analogous to those above.

α-Amino 412: ¹ H NMR (400 MHz, CD₃ OD) δ: 3.83 (m, 1H), 3.00 (br m, 1H),1.04 (s, 3H), 0.96 (d, J=6 Hz, 3H), 0.74 (s, 3H); IR (KBr, cm⁻¹): 3413,2942, 2061, 1710, 1594, 1460, 1377, 1167, 1074; MS(+FAB): 577.7 (M+1);Anal. calcd. for C₃₄ H₆₄ N₄ O₃ -4HCl-2.5H₂ O: C=53.19, H=9.58, N=7.30;Found: C=53.27, H=9.47, N=7.32.

β-Amino 413: ¹ H NMR (400 MHz, CD₃ OD) δ: 3.8 (m, 1H), 3.4 (m, 1H), 1.05(s, 3H), 0.96 (d, J=6 Hz, 3H), 0.73 (s, 3H); MS(+FAB): 577.7 (M+1).

Deoxycholic acid ethylenediamine 417 (α-amino isomer): ¹ H NMR (400 MHz,CD₃ OD) δ: 4.03 (m, 1H), 3.22 (br m, 1H), 1.03 (d, J=6 Hz, 3H), 1.00 (s,3H), 0.74 (s, 3H); IR (KBr, cm⁻¹): 2940, 1706, 1456, 1379, 1254, 1034;MS(+FAB): 435.4 (M+1); Anal. calcd. for C₂₆ H₄₆ N₂ O₃ -2HCl-2H₂ O:C=57.45, H=9.64, N=5.15; Found: C=57.32, H=9.22, N=5.13.

Deoxycholic acid spermine 449 (α-amino isomer): ¹ H NMR (400 MHz, CD₃OD) δ: 4.02 (m, 1H), 1.04 (d, J=6 Hz, 3H), 1.00 (s, 3H), 0.75 (s, 3H);IR (KBr, cm⁻¹): 2941, 1716, 1448, 1038; MS(+FAB): 577.4 (M+1); Anal.calcd. for C₃₄ H₆₄ N₄ O₃ -4HCl-1.5H₂ O: C=54.57, H=9.54, N=7.47; Found:C=54.31, H=8.71, N=7.80.

Example Q

Preparation of monoamine compounds 363 and 364: ##STR20##

Preparation of compound 1002: To a suspension of chromium trioxide (72.6g, 660 mmol) in dichloromethane (1000 ml) at -78° C., was added3,5-dimethylpyrazole (63.4 g, 660 mmol). After 20 minutes, cholesterylacetate (compound 1001, 24 g, 56 mmol) was added, and the mixture wasallowed to warm to room temperature slowly and stirred overnight. To thereaction mixture (0° C.) was added 5N sodium hydroxide solution (280ml), and the mixture was stirred for 1 hour. The organic phase waswashed with 2N HCl, water and brine. After removing the solvent, thecrude product was purified by chromatography (6 cm, gradient elutionwith 10% to 30% ethyl acetate in hexane) to afford starting material(6.78 g) and compound 1002 (12.78 g, 52%). ¹ H NMR (200 MHz, CDCl₃) δ:5.71 (s, 1H), 4.7 (br m, 1H), 2.5-1.0 (m, 27 H), 2.05 (s, 3H), 1.21 (s,3H), 0.92 (d, J=6.5 Hz, 3H), 0.86 (d, J=7 Hz, 6H), 0.68 (s, 3H).

Preparation of compound 1003: A solution of compound 1002 (14.32 g, 32.3mmol) in ethyl acetate (1.4 l) was purged with nitrogen, treated withplatinum(IV) oxide (263 mg), and hydrogen gas (atmospheric) for 3 hoursat room temperature. After filtration through Celite, the solution wasevaporated and purified by flash chromatography (6 cm, gradient elutionwith 0-20% ethyl acetate in hexane) to yield pure compound 1003 (10.86g, 76% yield). ¹ H NMR (200 MHz, CDCl₃) δ: 4.67 (br m, 1H), 2.4-1.0 (m,29H), 2.02 (s, 3H), 1.10 (s, 3H), 0.90 (d, J=6.5 Hz, 3H), 0.86 (d, J=6.5Hz, 6H), 0.65 (s, 3H); IR (KBr, cm⁻¹): 2950, 1730, 1707, 1471, 1373,1264, 1032; MS(+ES): 445.7 (M+1).

Preparation of compounds 1004 and 1005: To a solution of compound 1003(11.62 g, 26.1 mmol) in tetrahydrofuran (THF) (500 ml) was added 1MK-Selectride® (80 ml, 80 mmol) in THF at room temperature. After 5 hoursat 50° C., the reaction mixture was cooled in an-ice bath, and thentreated with 30% hydrogen peroxide (45 ml) and saturated ammoniumchloride solution (200 ml). The organic phase was separated and theaqueous phase was extracted with ether (3×100 ml), and the combinedorganic layers were washed with saturated sodium bicarbonate, ammoniumchloride and water. After drying, the crude product was purified bychromatography (6 cm, gradient elution with 0-30% ethyl acetate inhexane) to afford compound 1004 (10.95 g, 24.5 mmol), which wasdissolved in dichloromethane (200 ml) with dimethylaminopyridine (30.30g, 248 mmol) and treated with acetic anhydride (40 ml, 424 mmol) for 19hours. To this solution was added methanol (150 ml), and the solvent wasevaporated. The residue was dissolved in ethyl acetate, washed with 2Nhydrochloric acid solution (3×150 ml), water (100 ml), saturated sodiumcarbonate solution (3×100 ml) and brine (2×100 ml). The organic layerwas dried, evaporated, and purified by flash chromatography (6 cm,gradient elution with 0-20% ethyl acetate in hexane) to yield compound1005 (11.47 g, 90% yield). ¹ H NMR (200 MHz, CDCl₃) δ: 4.88 (m, 1H),4.71 (br m, 1H), 2.08 (s, 3H), 2.02 (s, 3H), 2.0-1.0 (m, 29H), 0.92-0.83(m, 12H), 0.64 (s, 3H) ; IR (KBr, cm ¹): 2954, 2867, 1730, 1466 1367,1257, 1025; Anal. calcd. for C₃₁ H₅₂ O₄ : C=76.18, H=10.72; Found:C=76.09, H=10.56.

Preparation of compound 1006: A solution of compound 1005 (10.85 g, 22.2mmol) and sodium cyanide (1.20 g, 24.4 mmol) in methanol (420 ml) wasstirred overnight at room temperature, and then refluxed for 10 hours.The solvent was evaporated, and the residue was dissolved indichloromethane and water, which was acidified with 2N hydrochloric acidsolution. After another dichloromethane extraction, the organic layerwas washed with brine, dried over magnesium sulfate, and evaporated toafford compound 1006 (9.22 g, 92% yield). ¹ H NMR (200 MHz, CDCl₃) δ:4.89 (m, 1H), 3.62 (br m, 1H), 2.07 (s, 3H), 2.0-1.0 (m, 29H), 0.90 (d,J=6 Hz, 3H), 0.87 (d, J=6.5 Hz, 6H), 0.82 (s, 3H), 0.64 (s, 3H); IR(KBr, cm⁻¹): 3446, 2935, 1735, 1469, 1375, 1245, 1042, 941; MS(+ES): 470(M+Na).

Preparation of compounds 1007, 1008 and 1009: To a solution of compound1006 (892 mg, 2.0 mmol) in anhydrous dichloromethane (20 ml) undernitrogen (-10° to -5° C.) was added triethylamine (3 ml, 22 mmol) andmethanesulfonyl chloride (0.40 ml, 5.2 mmol) in dichloromethane (4 ml).After 40 minutes, the mixture was poured into 1N hydrochloric acidsolution (100 ml), and the organic phase was separated. After extractingwith more dichloromethane (3×20 ml), the organic phase was washed with1N hydrochloric acid solution (30 ml), saturated sodium bicarbonatesolution (30 ml) and brine (2×30 ml). After drying over sodium sulfate,the solvent was evaporated to yield compound 1007, which was used forthe next step without purification.

Crude compound 1007 was dissolved in dimethylformamide (50 ml), treatedwith sodium azide (2.0 g, 31 mmol), and heated to 100° C. for 18 hours.After cooling, the reaction mixture was diluted with water (250 ml),extracted with dichloromethane (3×150 ml), washed with water (3×100 ml),dried (Na₂ SO₄), filtered, and evaporated to yield compound 1008, whichwas used in the next step without purification.

A solution of compound 1008 in anhydrous tetrahydrofuran (60 ml) wastreated with 1M lithium aluminum hydride (20 ml, 20 mmol) and heated toreflux for 5 hours. After cooling in an ice bath, to the mixture wasadded water (50 ml) and then 2M sodium hydroxide solution (200 ml). Theaqueous phase was extracted with dichloromethane (3×150 ml), followed bywashing with brine (2×100 ml) and water (50 ml). The dried organic layerwas evaporated to afford compound 1009, which was used withoutpurification in the next step.

Preparation of compounds 1363 and 1364: Crude compound 1009 wasdissolved in methanol (40 ml) and treated with 2N sodium hydroxidesolution (40 ml) at 80° C. for 12 hours. After evaporation, water wasadded (40 ml), followed by extraction with dichloromethane (3×60 ml).After washing with brine (3×50 ml), the organic layer was dried (Na₂SO₄), filtered and evaporated. Purification by flash chromatography(2-cm diameter, elution with 95:4.5:0.5dichloromethane:methanol:ammonium hydroxide) afforded compound 1363(slower eluting; ¹ H NMR (200 MHz, CDCl₃) δ: 3.82 (m, 1H), 3.19 (m, 1H),2.0-1.0 (m, 29H), 0.91 (d, J=6.5 Hz, 3H), 0.87 (d, J=6.5 Hz, 6H), 0.78(s, 3H), 0.65 (s, 3H); IR (KBr, cm⁻¹): 3362, 2931, 1575, 1467, 1382; MS(+FAB): 404.4 (M+1)) and compound 1364 (faster eluting; ¹ H NMR (200MHz, CDCl₃) δ: 3.4 (br m, 1H), 3.2 (m, 1H), 2.0-1.0 (m, 29H), 0.91 (d,J=6.5 Hz, 3H), 0.86 (d, J=6.5 Hz, 6H), 0.80 (s, 3H), 0.68 (s, 3H)). Eachcompound was dissolved in methanol, treated with excess 1N hydrogenchloride in ether, and evaporated to yield the hydrochloride salt.Compound 1363 (646 mg, 74% overall yield for 4 steps): Anal. calcd. forC₂₇ H₄₉ NO-HCl-0.5H₂ O: C=72.20, H=11.44, N=3.12; Found: C=72.40,H=11.44, N=3.26. Compound 1364 (50 mg, 6% overall yield for 4 steps):MS(+FAB): 404.4 (M+1); Anal. calcd. for C₂₇ H₄₉ NO-HCl-H₂ O: C=70.78,H=11.44, N=3.06; Found: C=71.02, H=11.33, N=3.35.

Example R

Preparation of compounds 388 and 387: ##STR21##

¹ H-NMR spectra were obtained on a Varian XL-200 (200 MHz) or a VarianUnity-500 (500 MHz) NMR spectrometer. Infrared spectra were recorded ona Perkin Elmer 298 spectrometer. Direct insertion probe (DIP) chemicalionization mass spectral data were obtained on a Hewlett Packard HP 5087GC-MS. Melting points were determined on a Thomas Hoover capillarymelting point apparatus and are uncorrected. Elemental analyses wereperformed by Quantitative Technologies Inc., Whitehouse, N.J. FAB massspectral data (low and high resolution) were obtained from M-Scan Inc.,West Chester, Pa.

5-Cholenic acid was obtained from Steraloids and used as received. Thefollowing reagents were purchased from Aldrich Chemical Company and wereused as received unless otherwise indicated: dihydropyran (distilledprior to use), p-toluenesulfonic acid, lithium aluminum hydride,t-butyldimethylsilyl chloride, imidazole, 3,5-dimethylpyrazole, platinum(IV) oxide, potassium tri-secbutylborohydride (K-Selectride®, 1M inTHF), hydrogen peroxide (30%), sodium hydride (60% in mineral oil),benzyl bromide (distilled prior to use), tetrabutylammonium fluoride (1Min THF), oxalyl chloride, diisopropylethylamine, dimethylsulfoxide(distilled prior to use), isopropylmagnesium chloride (2M in THF),magnesium bromide, sodium cyanoborohydride (1M in THF), 10% palladium oncarbon, and sulfur trioxide pyridine complex. THF and Et₂ O weredistilled from sodium/benzophenone ketyl. Pyridine was distilled fromKOH. Methylene chloride and pentane were distilled from CaH₂. DMF wasdistilled from Bao under reduced pressure. Methanol was dried over 3Åmolecular sieves prior to use. PPTS was prepared via the method ofMiyashita et al., J. Org. Chem. 42, 1977, 3772). Molecular sieves weredried in an oven (170° C.) overnight prior to use. Silica gel (EMScience Silica Gel 60, 230-400 mesh) was used for all flashchromatography.

Preparation of 3β-Tetrahydropyranyloxychol-5-en-24-oic acid24-tetrahydropyranyl ester (2001):

5-Cholenic acid (7.58 g, 20 mmol) was suspended in a solution of dry CH₂Cl₂ (300 ml). Distilled dihydropyran (19.0 ml, 200 mmol) was added,followed by a catalytic amount of pyridinium p-toluene sulfonate (1.1 g,4.0 mmol). The suspension was stirred at room temperature overnightunder argon. During this period of time, the steroid went into solution.The resultant solution was washed with a aqueous saturated NH₄ Clsolution (2x), aqueous saturated NaHCO₃ solution (2x), and aqueoussaturated NaCl solution. The organic layer was dried over anhydrousMgSO₄, filtered, and the solvent was removed in vacuo. The crude solidwas purified by flash chromatography (SiO₂, hexanes/EtOAc (10:1), givingcompound 2001 as a white solid (9.8 g, 18.5 mmol, 92%). ¹ H NMR (500MHz, CDCl₃) δ: 5.96 (brs, 1H, THP ester methine H), 5.37-5.32 (m, 1H,C-6 H), 4.72 (brs, 1H, THP ether methine H), 3.95-3.87 (m, 2H, THPCH-2), 3.71-3.64 (m, 1H, THP CH₂ O), 3.58-3.44 (m, 2H, THP CH₂ O & C-3H), 1.01 (s, 3H, C-19 H), 0.94 (d, J=6.3 Hz, 3H, C-21 H), 0.68 (s, 3H,C-18 H).

Preparation of 3β-Tetrahydropyranyloxychol-5-en-24-ol (2002):

Compound 2001 (16.1 g, 30 mmol) in dry tetrahydrofuran (THF, 150 ml) wasadded to a suspension of LiAlH₄ (5.5 g, 145 mmol) in dry THF (200 ml).The suspension was stirred at 0° C. with a mechanical stirrer underargon overnight. The resultant gray slurry was quenched with EtOAc,followed by aqueous saturated Na₂ SO₄ solution. During the addition ofthe Na₂ SO₄ solution, a white precipitate formed and the solution becameclear. Anhydrous Na₂ SO₄ was added, the mixture was stirred for 15minutes, and then filtered. The filter cake was washed well with ethylacetate and the filtrate was concentrated in vacuo. The resulting solidwas purified by flash chromatography (SiO₂, hexanes:EtOAc 5:1) givingcompound 2002 as a white solid (12.3 g, 27.7 mmol, 92%). ¹ H NMR (500MHz, CDCl₃) δ5.37-5.32 (m, 1H, C-6 H), 4.72 (brs, 1H, THP methine H),3.95-3.88 (m, 1H, THP CH₂ O), 3.62-3.47 (m, 4H, THP CH₂ O & C-3 H & C-24H), 1.01 (s, 3H, C-19 H), 0.93 (d, J=6.6 Hz, C-21 H), 0.68 (s, 3H, C-18H); IR (CHCl₃) 3610, 2900 cm⁻¹ ; MS (CI/isobutane) m/z 445 (M+1, 2%),343 (M+1-THPOH, 100%); m.p. 130°-131° C.

Preparation of24-t-Butyldimethylsilyloxy-3β-tetrahydropyranyloxychol-5-ene (2003):

Compound 2002 (7.6 g, 17 mmol) in dry CH₂ Cl₂ (300 ml) was treated witha solution of t-butyldimethylsilylchloride (TBDMSCl, 1.0M) and imidazole(0.5M) in dry CH₂ CO₂ (38.0 ml, 38.0 mmol TBDMSCl). The solution wasstirred at room temperature under argon overnight. The resultantsolution was poured into an aqueous saturated NaHCO₃ solution and themixture extracted with CH₂ CO₂ (3x). The combined organic layers werewashed with saturated sodium chloride, dried over anhydrous MgSO₄,filtered, and the solvent removed in vacuo. The resultant solid waspurified by flash chromatography (SiO₂, hexanes:EtOAc gradient from 20:1to 5:1) giving compound 2003 (9.4 g, 17 mmol, 98%). ¹ H NMR (500 MHz,CDCl₃) δ: 5.38-5.32 (m, 1H, C-6 H), 4.72 (brs, 1H, THP methine H),3.95-3.88 (m, 1H, THP CH₂ O), 3.60-3.46 (m, 4H THP CH₂ O & C-3 H & C-24H), 1.01 (s, 3H, C-19 H), 0.93 (d, J=6.6 Hz, C-21 H), 0.89 (s, 9H,t-Bu), 0.67 (s, 3H, C-18 H), 0.05 (s, 6H, TBDMS CH₃); IR (CHCl₃) 2900cm⁻¹ ; MS (CI/isobutane) m/z 559 (M+1, 1%), 474 (M+1-THP, 12%), 457(M+1-THPOH, 18%), 343 (M+1-THP-TBDMSOH, 6%), 325 (M+1-THPOH-TBDMSOH,100%); m.p. 116°-118° C; Anal. calcd. for C₃₅ H₆₂ O₃ Si: C=75.21,H=11.18; Found: C=75.37, H=11.24.

Preparation of24-t-Butyldimethylsilyloxy-3β-tetrahydropyranyloxychol-5-en-7-one(2004):

Chromium trioxide (6.43 g, 64.4 mmol) was suspended in dry CH₂ Cl₂ (100ml). The mechanically stirred suspension under argon was cooled to -78°C. via a dry-ice/acetone bath. 3,5-Dimethylpyrazole (6.18 g, 64.4 mmol)was added to the suspension as a solid and the mixture was allowed tostir for 25 minutes at -78° C. to ensure complete formation of thecomplex. Compound 2003 (3.10 g, 5.37 mmol) was then added to the mixtureas a solid, and the reaction mixture was allowed to slowly warm to roomtemperature and stirred overnight. The mixture was then transferred to aone-neck 500 ml round-bottom flask and silica gel (flash grade) wasintroduced. The slurry was concentrated to a free-flowing solid whichwas introduced onto the top of a wet packed flash column (SiO₂) and theproduct was eluted with hexanes:ethyl acetate (gradient 30:1 to 15:1 to6:1 to 3:1). The desired product, compound 2004 (1.80 g, 59%) wasobtained as a white solid. ¹ H NMR (500 MHz, CDCl₃) δ: 5.65 & 5.63 (2S,1H, C-6 H), 4.70-4.64 (m, 1H, THP methine), 3.90-3.81 (m, 1H, THP CH 2),3.70-3.62 (m, 1H, C-3H or THP CH₂ O), 3.57 (t, J=6.6 Hz, 2H, C-24 H),3.52-3.46 (m, 1H, C-3 H or THP CH₂ O), 1.19 (s, 3H, C-19 H), 0.93 (d,J=6.3 Hz, C-21 H), 0.90 (s, 9H, t-butyl), 0.68 (s, 3H, C-18 H), 0.05 (s,6H, TBDMS CH₃); IR (CHCl₃) 2900, 1650 cm⁻¹ ; MS (CI/isobutane) m/z 573(M+1, 11%), 489 (M+1-THP, 100%); m.p. 118°-120° C.

Preparation of24-t-Butyldimethylsilyloxy-3β-tetrahydropyranyloxy-5α-cholan-7-one(2005):

Compound 2004; (1.0 g, 1.75 mmol) was dissolved in EtOAc (75 ml) andplatinum(IV) oxide (0.012 g, 0.049 mmol) was added. The mixture wasplaced on a hydrogenation apparatus (atmospheric). The set-up wasevacuated to remove the dissolved oxygen and then hydrogen wasintroduced. The evacuation and introduction of hydrogen process wasrepeated 2 times. The reaction was stirred under hydrogen at atmosphericpressure for 2.5 hours. The reaction mixture was filtered through Celiteand concentrated in vacuo. The crude product was purified by flashchromatography (SiO₂, hexanes:EtOAc gradient starting with 20:1) givingcompound 2005 as a white solid (0.70 g, 71%).24-t-Butyldimethylsilyloxy-3β-tetrahydropyranyloxy-5α-cholan-7.beta.-olwas obtained as a by-product (21%). (Note: This by-product could beconverted to the desired ketone 2005 with Collin's reagent in 64%yield.) Compound 2005: ¹ H NMR (500 MHz, CDCl₃) δ: 4.73-4.66 (m, 1H, THPmethine H), 3.95-3.85 (m, 1H, THP CH₂ O), 3.66-3.52 (m, 3H, THP CH₂ O &C-24 H), 3.50-3.45 (M, 1H, C-3 H), 1.08 (s, 3H, C-19 H), 0.91 (d, J=6.6Hz, C-21 H), 0.89 (s, 9H, t-Bu), 0.64 (s, 3H, C-18 H), 0.04 (s, 6H,TBDMS CH₃); IR (CHCl₃) 2900, 1685 cm⁻¹ ; MS (Ci/isobutane) m/z 575 (M+1,85%), 491 (M+1-THP, 100%); m.p. 166°-170° C.; Anal. calcd. for C₃₅ H₆₂O₄ Si: C=73.12, H=10.87; Found: C=72.88, H=10.78.

Preparation of24-t-Butyldimethyulsilyloxy-3β-tetrahydropyranyloxy-5α-cholan-7α-ol(2006):

K-Selectride® (potassium tri-sec-butylborohydride) (8.9 ml, 1M in THF,8.9 mmol) was added dropwise via syringe to a solution of ketone 2005(1.7 g, 3.0 mmol) in dry THF (50 ml) at room temperature under argon.The reaction mixture was heated to 50° C. in an oil bath and stirred for5 hours. The mixture was allowed to cool to room temperature and thenquenched by adding 30% H₂ O₂ dropwise until the evolution of gas ceased.Saturated aqueous NH₄ Cl solution was added and the aqueous solution wasextracted (3x) with Et₂ O. The combined organic extracts were washedwith aqueous saturated NaHCO₃ solution (2x), distilled H₂ O (2x), andaqueous saturated NaCl solution, dried over anhydrous MgSO₄, filtered,and the solvent was removed in vacuo. The crude product was purified byflash chromatography (SiO₂, hexanes:EtOAc 10:1) giving alcohol 2006 as awhite solid (1.6 g, 94%). ¹ H NMR (500 MHz, CDCl₃) δ: 4.73-4.66 (m, 1H,THP methine H), 3.95-3.85 (m, 1H, THP CH₂ O), 3.82 (s, 1H, C-7 H),3.66-3.52 (m, 3H, THP CH₂ O & C-24 H), 3.50-3.45 (m, 1H, C-3 H), 1.08(s, 3H, C-19 H), 0.91 (d, J=6.6 Hz, C-21 H), 0.89 (s, 9H, t-Bu), 0.64(s, 3H, C-18 H), 0.04 (s, 6H, TBDMS CH₃); MS (CI/isobutane) m/z 577(M+1, 5%), 493 (M+1-THP, 22%), 475 (M+1-THPOH, 26%), 458 (M+1-THPOH-H₂O, 38%), 343 (M+1-THPOH-TBDMSOH, 80%), 325 (M+1-THPOH-TBDMSOH-H₂ O,100%); IR (CHCl₃) 3430, 2860 cm⁻¹ ; m.p. 130°-133° C.; Anal. calcd. forC₃₅ H₆₄ O₄ Si: C=72.86, H=11.18; Found: C=72.69, H=11.32.

Preparation of7α-Benzyloxy-24-t-butyldimethylsilyloxy-3β-tetrahydropyranyloxy-5α-cholane(2007):

A flame-dried round-bottom flask with stirring bar was charged withsodium hydride (60% in mineral oil, 28 mg, 0.69 mmol), equipped with aseptum and a gas-needle inlet and flushed with argon. The mineral oilwas removed by washing (3x) with dry pentane, and the pentane wasremoved to provide the sodium hydride as a powder. Dry DMF (2.0 ml) wasadded. A solution of alcohol 2006 (40 mg, 0.069 mmol) in dry THF (2 ml)was added dropwise via syringe. The reaction mixture was stirredovernight and then heated to 40° C. in an oil bath over a 20-minuteperiod. Freshly distilled benzyl bromide (0.165 ml, 1.38 mmol) was addeddropwise, and the reaction mixture was stirred at 40° C. for 10 hours.The reaction was allowed to cool to room temperature, and the solventwas removed under reduced pressure. The flask was placed under vacuumovernight to remove any residual DMF. The crude material was purified byflash chromatography (SiO₂, hexanes:EtOAc 50:1) giving compound 2007 asa white solid (40 mg, 0.060 mmol, 87%). A gradient of increasing EtOAcconcentration provided other components, including the 7α-formate (1 mg,1%) as well as recovered starting material (3 mg, 8%). Compound 2007: ¹H NMR (500 MHz, CDCl₃) δ: 7.35-7.20 (m, 5H, benzyl Ar-CH₂), 4.73-4.66(m, 1H, methine H), 4.535 (d, J=12.0 Hz, 1/2H, benzyl-CH₂), 4.53 (d,J=12.0 Hz, 1/2H, benzyl-CH₂), 4.26 (d, J=12.2 Hz, 1/2H, benzyl-CH₂),4.245 (d, J 11.8 Hz, 1/2H, benzyl-CH₂), 3.95-3.85 (m, 1H, THP CH₂ O),3.66-3.52 (m, 3H, THP CH₂ O & C-24 H), 3.50-3.45 (m, 1H, C-3 H), 1.08(s, 3H, C-19 H), 0.91 (d, J=6.6 Hz, C-21 H), 0.89 (s, 9H, t-Bu), 0.64(s, 3H, C-18 H), 0.04 (s, 6H, TBDMS CH₃); MS (CI/isobutane) m/z 668(M+1, 6%), 584 (M+1-THP, 18%), 475 (M+1-THPOH, 30%), 457(M+1-THPOH-HOBn, 58%), 343 (M+1-THP-HOBn-TBDMSOH, 100%), 325(M+1-THPOH-TBDMSOH-HOBn, 83%).

Preparation of 7α-Benzyloxy-3β-tetrahydropyranyloxy-5α-cholan-24-ol(2008):

Compound 2007 (0.0527 g, 0.079 mmol) in anhydrous THF (4 ml) under Arwas treated with tetrabutylammonium fluoride (TBAF) (0.237 ml, 1M inTHF, 0.237 mmol). The solution was stirred until no starting materialremained by TLC. The solvent was removed in vacuo, the residue taken upin 5 ml CH₂ Cl₂, washed with 5 ml aqueous saturated NaHCO₃ solution, andthe aqueous layer was extracted 2x with 5 ml CH₂ Cl₂. The combinedorganic layers were dried over anhydrous MgSO₄, filtered, and thesolvent removed in vacuo. Flash chromatography (SiO₂, 8:1 hexanes:EtOAc)gave compound 2008 (0.0395 g, 90%) as a white solid foam. ¹ H NMR (500MHz, CDCl₃) δ: 7.35-7.34 (m, 5H, benzyl Ar-H), 4.71-4.69 (m, 1H, THPether methine H), 4.585 (d, J=11.8 Hz, 1/2H, benzyl-CH₂), 4.58 (d,J=11.8 Hz, 1/2H, benzyl-CH₂), 4.315 (d, J=12.0 Hz, 1/2H, benzyl-CH₂),4.29 (d, J=12.0 Hz, 1/2H, benzyl-CH₂), 3.94-3.90 (m, 1H, THP OCH₂),3.62-3.58 (m, 3H, C-24 H & THP OCH₂), 3.50-3.48 (m, 1H, C-3 H), 3.45 (s,1H, Z-H), 0.92 (d, J=6.6 Hz, 3H, C-21 H), 0.81 (s, 3H, C-19 H), 0.63 (s,3H, C-18 H) (Note: product is a mixture of diastereomers); IR (CHCl₃)3600, 2900 cm⁻¹ ; MS (CI/isobutane) m/z 554 (M+1, 2%), 361(M+1-THP-HOBn, 42%), 343 (M+1-THP-HPBn, H₂ O, 100%); m.p. 52°-56° C.;Anal. calcd. for C₃₆ H₅₆ O₄ : C=78.21, H=10.21; Found: C=77.93, H=10.39.

Preparation of 7α-Benzyloxy-3β-tetrahydropyranyloxy-5α-cholan-24-al(2009):

DMSO (0.01 ml, 0.14 mmol) in CH₂ Cl₂ (0.1 ml) was added dropwise to astirred solution of oxalyl chloride (0.008 ml, 0.0917 mmol) in anhydrousCH₂ Cl₂ (2 ml) at -78° C. under anhydrous conditions (drying tube). Thissolution was stirred at -78° C. for 15 minutes. Steroid 2008 (0.0234 g,0.0423 mmol) in dry CH₂ Cl₂ (0.5 ml) was then added dropwise and thesolution stirred for 40 minutes at -78° C. Diisopropylethylamine (DIPEA)(0.08 ml, 0.458 mmol) was added and the solution allowed to warm to 0°C. with stirring over a 30-minute period. Aqueous saturated NaHCO₃solution (5 ml) was added and the solution extracted 3x with 5 ml CH₂Cl₂. The combined organic extracts were washed 2x with 5 ml of aqueoussaturated NaCl solution, dried over anhydrous MgSO₄, filtered, and thesolvent removed in vacuo. Flash chromatography (SiO₂, 10:1hexanes:EtOAc) gave compound 2009 (0.0226 g, 97%) as a white solid foam.¹ H NMR (500 MHz, CDCl₃) δ: 9.76 (s, 1H, C-24 H), 7.35-7.34 (m, 5H,benzyl Ar-H), 4.71-4.69 (m, 1H, THP ether methine H), 4.59 (d, J=11.8Hz, 1/2H, benzyl-CH₂), 4.585 (d, J=11.8 Hz, 1/2H, benzyl-CH₂), 4.30 (d,J=12.0 Hz, 1/2H, benzyl-CH₂), 4.29 (d, J=12.0 Hz, 1/2H, benzyl-CH₂),3.95-3.89 (m, 1H, THP OCH₂), 3.63-3.58 (m, 3H, C-24 H & THP OCH₂),3.50-3.47 (m, 1H, C-3 H), 3.45 (s, 1H, 7-H), 2.49-2.42 (m, 1H, C-23H),2.37-2.31 (m, 1H, C-23 H), 0.958 (d, J=6.5 Hz, 3H, C-21 H), 0.81 (s, 3H,C-19 H), 0.63 (s, 3H, C-18 H) (Note: product is a mixture ofdiastereomers); IR (CHCl₃) 2900, 1700 cm⁻¹ ; MS (CI/isobutane) m/z 552(M+1, 0.4%), 465 (M+1-THP, 3%), 449 (M+1-THPO, 14%), 375 (M+1-THP-Bn,7%), 359 (M+1-THP-HOBn, 68%), 341 (M+1-THP-HOBn-H₂ O, 100%); m.p.50°-54° C.; Anal. calcd. for C₃₆ H₅₄ O₄ : C=78.50, H=9.88; Found:C=78.11, H=10.04.

Preparation of 7α-Benzyloxy-3β-tetrahydropyranyloxycholestan-24ε-ol(2010):

A solution of compound 2009 (0.374 g, 0.679 mmol) in anhydrous THF (10ml) under argon was treated with isopropylmagnesium chloride (2 ml, 2Min THF, 5.43 mmol) at room temperature. The reaction was stirred untilno starting material remained by TLC. Aqueous NH₄ Cl solution (10%, 15ml) was added to quench the reaction and the THF was removed in vacuo.Distilled H₂ O (5 ml) was added and the solution extracted 3x with 15 mlCH₂ Cl₂. The combined organic layers were washed with aqueous saturatedNaCl solution (15 ml), dried over anhydrous MgSO₄, filtered and thesolvent removed in vacuo. Flash chromatography (SiO₂, 12:1hexanes:EtOAc) gave compound 2010 (0.3117 g, 77%) as a white foam. ¹ HNMR (500 MHz, CDCl₃) δ: 7.35-7.34 (m, 5H, benzyl Ar-H), 4.71-4.69 (m,1H, THP ether methine H), 4.585 (d, J=11.9 Hz, 1H, benzyl-CH₂), 4.31 (d,J=12.0 Hz, 1/2H, benzyl-CH₂), 4.295 (d, J=12.0 Hz, 1/2H, benzyl-CH₂),3.94-3.91 (m, 1H, THP, OCH₂), 3.62-3.58 (m, 1H, THP OCH₂), 3.50-3.48 (m,1H, C-3 H), 3.45 (s, 1H, C-7 H), 3.32-3.31 (m, 1H, C-24 H), 0.81 (s, 3H,C-19 H), 0.63 (d J=2.4 Hz, 3H, C-18 H) (Note: product is a mixture ofdiastereomers); IR (CHCl₃) 3605, 2900 cm⁻¹ ; MS (CI/isobutane) m/z 595(M+1, 10%), 401 (M+1-THP-Bn-H₂ O, 25%), 385 (M+1-THP-HOBn, H₂ O, 100%);m.p. 55°-59° C.; Anal. calcd. for C₃₉ H₆₂ O₄ : C=78.74, H=10.50; Found:C=78.65, H=10.54.

Preparation of7α-Benzyloxy-24ε-t-butyldimethylsilyloxy-3β-tetrahydropyranyl-oxycholestane(2011):

Compound 2010 (0.050 g, 0.084 mmol) in dry CH₂ Cl₂ (1 ml) was treatedwith a solution of t-butyldimethylsilylchloride (TBDMSCl, 0.5 M) andimidazole (1.0M) in dry CH₂ Cl₂ (0.80 ml, 0.40 mmol TBDMSCl). Thereaction was stirred at room temperature under argon for 24 hours.Aqueous saturated NaHCO₃ solution (5 ml) was added and the solutionextracted 3x with 10 ml CH₂ Cl₂. The combined organic layers were washedwith 10 ml aqueous saturated NaCl solution and dried over anhydrous Na₂SO₄. Filtration and removal of solvent in vacuo followed by flashchromatography (SiO₂, 20:1 hexanes:EtOAc) gave the desired product 2011(0.057 g, 96%) as a white solid. ¹ H NMR (500 MHz, CDCl₃) δ: 7.35-7.34(m, 5H, benzyl Ar-H), 4.70-4.69 (m, 1H, THP ether methine H), 4.59 (d,J=12.0 Hz, 1H, benzyl-CH₂), 4.315 (d, J=12.0 Hz, 1/2H, benzyl-CH₂), 4.31(d, J=12.0 Hz, 1/2H, benzyl-CH₂), 3.943.91 (m, 1H, THP OCH₂), 3.62-3.58(m, 1H, THP OCH₂), 3.50-3.48 (m, 1H, C-3 H), 3.45 (s, 1H, C-7 H),3.37-3.35 (m, 1H, C-24 H), 0.89 (d, J=1 Hz, 9H, SiC(CH₃)₃,diastereomeric at C-24), 0.81 (s, 3H, C-19 H), 0.62 (s, 3H, C-18 H),0.04 & 0.03 (2s, 6H, Si(CH₃)₂, diastereotopic and/or diastereomeric)(Note: product is a mixture of diastereomers); IR (CHCl₃) 2900 cm ; MS(CI/isobutane) m/z 709 (M+1, 20%), 367 (M+1-THPOH-HOBn-TBDMSOH, 100%);m.p. 52°-58° C.; Anal. calcd. for C₄₅ H₇₆ O₄ S: C=76.21, H=10.80; Found:C=76.11, H=10.81.

Preparation of 7α-Benzyloxy-24ε-t-butyldimethylsilyloxycholestan-3β-ol(2012):

Compound 2011 (0.057 g, 0.0803 mmol) was dissolved in dry Et₂ O (3 ml)under argon. MgBr₂ (0.142 g, 0.771 mmol) was added quickly as a solidand the reaction was stirred until no starting material remained by TLC.H₂ O (10 ml) was added and the mixture was extracted 3x with 10 ml Et₂O. The combined organic layers were dried over anhydrous MgSO₄, filteredand the solvent removed in vacuo. Flash chromatography (SiO₂, 7:1hexanes:EtOAc) gave compound 2012 (0.0493 g, 98%) as a white foam. ¹ HNMR (500 MHz, CDCl₃) δ: 7.36-7.35 (m, 5H, benzyl-Ar H), 4.59 (d, J=12.0Hz, 1H, benzyl-CH₂), 4.34 (d, J=12.0 Hz, 1H, benzyl-CH₂), 3.65-3.60 (m,1H, C-3 H), 3.475 (d, J=2.4 Hz, 1H, C-7 H), 3.40-3.36 (m, 1H, C-24 H),0.91 (d, J=0.9 Hz, 9H, SiC(CH₃)₃, diastereomeric at C-24), 0.82 (s, 3H,C-19 H), 0.65 (s, 3H, C-18 H), 0.05 & 0.04 (s, 6H, Si(CH₃)₂,diastereotopic and/or diastereomeric) (Note: product is a mixture ofdiastereomers); IR (CHCl₃) 3600, 2900 cm⁻¹ ; MS (CI/isobutane) m/z 624(M+1, 3%), 501 (M+1-OTHP, 6%), 385 (M+1-OTHP-TBDMS, 68%), 367(M+1-THPOH-TBDMSOH, 100%); m.p. 55°-58° C.; Anal. calcd. for C₄₀ H₆₈ O₃Si: C=76.86, H=10.97; Found: C=76.69, H=10.87.

Preparation of 7α-Benzyloxy-24ε-t-butyldimethylsilyloxycholest-3-one(2013a) and 7α-Benzoyloxy-24ε-tbutyldimethylsilyloxycholestan-3-one(2013b):

A solution of compound 2012 (0.229 g, 0.3664 mmol) in dry CH₂ Cl₂ (30ml) was treated with Collin's reagent (0.385 g, 1.49 mmol). The mixturewas stirred at room temperature overnight under argon. At this time nostarting material remained by TLC. Celite was added and the mixture wasstirred for 20 minutes and then filtered through a pad of Celite. Thecake was rinsed well with CH₂ Cl₂. The solvent was removed in vacuo.Flash chromatography (SiO₂, 20:1 hexanes:EtOAc) gave the desired product2013a (0.198 g, 87%) as a white solid along with the 7α-benzoate 2013b(0.015 g, 6.4%) as a white foam. Note: If the reaction was run at higherconcentration, a higher yield of the benzoate was obtained. Compound2013a: ¹ H NMR (500 MHz, CDCl₃) δ: 7.35-7.27 (m, 5H, benzyl Ar-H), 4.55(d, J=11.7 Hz, 1H, benzyl-CH₂), 4.32 (d, J=11.7 Hz, benzyl-CH₂), 3.495(d, J=2.0 Hz, 1H, C-7 H), 3.38-3.35 (m, 1H, C-24 H), 1.02 (s, 3H, C-19H), 0.90 (d, J=0.8 Hz, 9H, SiC(CH₃)₃, diastereomeric at C-24), 0.67 (s,3H, C-18 H), 0.04 & 0.03 (s, 6H, Si(CH₃)₃, diastereotopic and/ordiastereomeric) (Note: product is a mixture of diastereomers); IR(CHCl₃) 2900, 1690 cm⁻¹ ; MS (CI/isobutane) m/z 624 (M+1, 50%), 534(M+1-Bn, 7%), 518 (M+1-OBn, 36%), 492 (M+1-HOSi(Me)₂ t-Bu, 28%), 383(M+1-C₁₄ H₃₀ OSi, 100%). Compound 2013b: ¹ H NMR (500 MHz, CDCl₃) δ:8.03 (d, J=7.3 Hz, 2H, benzoate-Ar H), 7.59 (t, J=7.4 Hz, 1H, Ar H),7.48 (t, J=7.7 Hz, 2H, Ar H), 5.20 (br s, 1H, C-7 H), 3.35-3.31 (m, 1H,C-24 H), 1.08 (s, 3H, C-19 H), 0.86 (d, J=3.7, 9H, SiC(CH₃)₃), 0.71 (s,3H, C-18 H) (Note: product is a mixture of diastereomers); IR (CHCl₃)2900, 1690 cm⁻¹ ; MS (CI/isobutane) m/z 637 (M+1, 3%), 516 (M+1-OBz,16%), 382 (M+1-OBz-TBDMSOH, 100%); m.p. 62°-65° C.

Preparation of7α-Benzyloxy-3ε-(5,10-di-t-butoxycarbonyl-1,5,10-triazadecyl)-24ε-t-butyldimethylsilyloxycholestane(2014a):

A mixture of compound 2013a (0.07 g, 0.11 mmol), approximately 2equivalents of amino compound 301 (based on 60% yield for the reductionof compound 2018 to compound 301), and 3 Å molecular sieves (0.5 g) inMeOH (6 ml, dried over 3 Å sieves) was stirred for 12 hours at roomtemperature under argon. NaCNBH₃ (0.33 ml, 1M in THF, 0.33 mmol) wasadded and the solution stirred for 24 hours at room temperature underargon. The mixture was filtered through Celite and the cake was washedwell with MeOH and CH₂ Cl₂ and the solvents were removed in vacuo. Theresidue was dissolved in CH₂ Cl₂ (10 ml), washed 2x with 5 ml H₂ O madebasic with aqueous NaOH solution (5%), and washed 2x with 5 ml aqueoussaturated NaCl solution. The combined aqueous layers were back-extractedwith CH₂ Cl₂, and the combined organic layers were dried over anhydrousMgSO₄. Filtration, removal of the solvent in vacuo, and flashchromatography (SiO₂) gradient of increasing polarity from 2% MeOH inCH₂ Cl₂ to 10% MeOH in CH₂ Cl₂) gave the desired product 2014a (0.07 g,66%) and a more polar product, compound 2014b, which is missing onet-BOC group and is contaminated with excess amine. Compound 2014a: ¹ HNMR (500 MHz, CDCl₃) δ: 7.36-7.28 (m, H, benzyl-Ar H), 4.63 (d, J 12.0Hz, 1/2H, benzyl-CH₂), 4.58 (d, J=12.0 Hz, 1/2H, benzyl-CH₂), 4.33 (t,J=1.25 Hz, 1H, benzyl-CH₂), 3.49 (s, 1H, C-7 H), 3.46-3.14 (m, 8H,N(BOC)CH₂ & C-24 H), 2.91-2.86 (m, 2H, NCH₂), 1.47-1.41 (m, 18H,2xCOC(CH₃)₃), 0.90 (s, 9H, SiC(CH₃)₃), 0.84 (s, 3H, C-19 H), 0.64 (s,3H, C-18 H), 0.05 & 0.04 (s, 6H, Si(CH₃)₂, diastereotopic and/ordiastereomeric) (Note: This product is a mixture of diastereomers).

Preparation of 7α-Benzyloxy-3β-(1,5,10-triazadecyl)cholestan-24ε-ol(2015β) and 7α-benzyloxy-3α-(1,5,10-triazadecyl)cholestan-24ε-ol(2015α):

TFA (1.8 ml, 24 mmol) was added to a solution of compound 2014a (0.386g, 0.4 mmol) in CHCl₃ (15 ml) at room temperature. The reaction wasstirred until no starting material remained by TLC. The solvent wasremoved in vacuo and the residue purified by preparative TLC (SiO₂, 2000μm, 6:3:1 CH₂ Cl₂ :MEOH:NH₄ OH, R_(f) =0.46) to give the desired 3βproduct 2015β (0.122 g, 48%) and the 3α isomer 2015α (0.109 g, 43%).Compound 2014b could be treated with TFA under the same conditions togive compounds 2015α and 2015β. Compound 2015β: ¹ H NMR (500 MHz, CD₃OD) δ: 7.32-7.35 (m, 5H, benzyl-Ar H), 4.57 (d, J=11.7 Hz, 1H,benzyl-CH₂), 4.31 (d, J=11.7 Hz, 1H, benzyl-CH₂), 3.52 (s, 1H, C-7 H)3.22-3.21 (m, 2H, C-24 H & NCH₂) 2.86 (t, J=7.1 Hz, 2H, NCH₂), 2.81 (t,J=6.6 Hz, 2H, NCH₂), 2.74 (t, J=7.0 Hz, 2H, NCH₂), 2.67 (t, J=6.3 Hz,2H, NCH₂), 0.85 (s, 3H, C-19 H), 0.683 (s, 1.5H, C-18, diastereomeric atC-34), 0.678 (s, 1.5H, C-18, diastereomeric at C-24); MS (pos. FAB) m/z638.6 (M+1, 100%). Compound 2015a: ¹ H NMR (500 MHz, CD₃ OD) δ:7.35-7.22 (m, 5H, benzyl-Ar H), 4.61 (d, J=11.4 Hz, 1H, benzyl-CH₂),4.28 (d, J=11.5 Hz, 1H, benzyl-CH₂), 3.53 (s, 1H, C-7 H), 3.43 (s, 1H,C-3 H), 3.24-3.20 (m, 2H, C-24 H & NCH₂), 3.11 (t, J=7.1 Hz, 2H, NCH₂),3.08-3.02 (m, 2H, NCH₂), 2.96 (t, J=6.9 Hz, 2H, NCH₂), 0.85 (s, 3H, C-19H), 0.691 (s, 1.5H, C-18, diastereomeric at C-24), 0.686 (s, 1.5H, C-18,diastereomeric at C-24); MS (pos. FAB) m/z 638.6 (M+1, 100%).

Preparation of 3α-(1,5,10-Triazadecyl)cholesta-7α,24ε-diol (2016):

To a solution of compound 2015β (0.0128 g, 0.02 mmol) in absolute EtOH(8 ml) was added a catalytic amount of 10% Pd/C and 2 drops ofconcentrated hydrochloric acid. The mixture was placed on a Parrhydrogenation apparatus and shaken under 55 psi (H₂) for 24 hours. Thesolution was filtered through a pad of Celite, and the cake was washedwell with EtOH and MeOH, and the solvents removed in vacuo. The desiredproduct 2016 (0.0074 g, 68%) was obtained. If the product was pure byTLC, it was used without further purification. If impurities wereobserved by TLC, the material was purified by flash chromatography(SiO₂, 15:4:1 CH₂ C₂ :MeOH:NH₄ OH). ¹ H NMR (500 MHz, CD₃ OD) δ: 3.79(s, 1H, C-7 H) 3.22-3.13 (m, 6H, 2xCH₂ N & C-24 H & C-3 H), 3.09 (t,J=7.4, 2H, CH₂ N), 2.99 (t, J=7.3 Hz, 2H, CH₂ N), 0.87 (s, 3H, C-19 H),0.694 (s, 1.5H, C-18 H, diastereomeric at C-24), 0.691 (s, 1.5H, C-18 H,diastereomeric at C-24).

Preparation of squalamine (compound 1256):

Compound 2016 (0.0176 g, 0.032 mmol) was dissolved in a solution ofconcentrated hydrochloric acid in MeOH (1 ml concentrated hydrochloricacid in 10 ml MeOH). The solution was stirred for 15 minutes and thesolvent removed in vacuo. To the crude, dried product was added SO₃-pyridine complex (0.010 g, 0.064 mmol) and the flask was flushed withargon. Dry pyridine (1 ml) was added, the solution was warmed to 80° C.in an oil bath and stirred for 2 hours. MeOH (2 ml) was added. The flaskwas removed from the oil bath and the mixture was stirred for 15minutes. The solvent was removed in vacuo, and the residue wasresuspended in MeOH and filtered through a pad of Celite. The cake waswashed well with MeOH. Flash chromatography (SiO₂, 12:4:1 CH₂ Cl₂:MEOH:NH₄ OH) gave the desired product 1256 (0.0113 g, 56%) as a whitesolid. ¹ H NMR (500 MHz, CD₃ OD) δ: 4.13-4.10 (m, 1H, C-24 H), 3.79 (s,1H, C-7 H), 3.22-3.10 (m, 5H CH₂ N), 3.08 (t, J=6.7 Hz, 2H, CH₂ N), 2.98(t, J=6.8 Hz, 2H, CH₂ N), 0.87 (s, 3H, C-19 H), 0.70 (s, 3H, C-18 H); MS(pos. FAB) m/z 628.4 (M+1, 57%), 548.5 (M+1-SO₃, 23%), 530.5 (M+1-H₂S0₄, 100%); high resolution MS (pos. FAB) m/z 628.4669 (calcd.:628.4723).

Preparation of 5,10-Di-t-butoxycarbonyl-1,5,10-triazadecane (301):

Nitrile 2018 (0.0624 g, 0.181 mmol) in dry Et₂ O (0.30 ml) was added toa suspension of LiAlH₄ (0.024 g, 0.63 mmol) in dry diethyl ether (1 ml)at 0° C. The mixture was stirred at 0° C. for 30 minutes. Aqueous NaOHsolution (1M) was added to quench excess LiAlH₄, and the resulting whitesuspension was filtered through a pad of Celite. The cake was washedwell with Et₂ O, and the combined organic layers were washed with H₂ O.The H layer was extracted with Et₂ O and the combined ether layers werewashed with aqueous saturated NaCl solution, dried over anhydrous MgSO₄,filtered and the solvent removed in vacuo. The ¹ H NMR spectrum (500MHz) of the crude product 301 (0.056 g, 88%) matched that reported inthe literature (Tetrahedron 46, 1990, 3267-3286), and the material wasused crude.

Preparation of7α-Benzoyloxy-3ε-(5,10-di-t-butoxycarbonyl-1,5,10-triazadecyl)-24ε-t-butyldimethylsilyloxycholestane(2020):

Compound 2013b (0.110 g, 0.1726 mmol) was converted to compound 2020(0.166 g, 99%) using the previously described procedure for theconversion of compound 2013a to compound 2014a. ¹ H NMR (500 MHz, CDCl₃)δ: 8.19 (d, J=7.6 Hz, 1/2H, benzoate-Ar H), 8.05 (d, J=7.4 Hz, 3/2H,benzoate-Ar H), 7.61-7.58 (m, 1H, benzoate-Ar H), 7.55-7.45 (m, 2H,benzyl-Ar H), 5.23 (s, 1/4H, C-7 H), 5.16 (s, 3/4H, C-7 H), 4.78-4.64(m, 1H), 3.40-3.22 (m, 2H), 3.20-3.06 (m, 3H, NCH₂), 2.98-2.8 (m, 4H,NCH₂), 0.673 (s, 1.5H, C-18, diastereomeric at C-24), 0.667 (s, 1.5H,C-18, diastereomeric at C-24).

Preparation of 7α-Benzoyloxy-3α-(1,5,10-triazadecyl)cholestan-24ε-ol(2021α) and 7α-benzoyloxy-3β-(1,5,10-triazadecyl)cholestan-24β-ol(2021β):

Compound 2020 (0.166 g, 0.1717 mmol) was converted to compounds 2021αand 2021β (quantitative yield of a 1:1 mixture of the 3α and 3βproducts) in the same manner as described previously for the conversionof compound 2014a to compounds 2015α and 2015β. Compound 2021β: ¹ H NMR(500 MHz, CD₃ OD) δ: 8.01 (d, J=8.3 Hz, 2H, benzoate-Ar H), 7.61-7.59(m, 1H, benzoate-Ar H), 7.51-7.45 (m, 2H, benzyl-Ar H), 5.14 (s, 1H, C-7H), 3.20-3.15 (m, 1H), 2.90-2.75 (m, 4H, NCH₂), 2.72 (t, J=6.9 Hz, 2H,NCH₂), 2.65 (t, J=6.7 Hz, 2H, NCH₂), 0.85 (s, 3H, C-19 H), 0.726 (s,1.5H, C-18, diastereomeric at C-24), 0.723 (s, 1.5H, C-18,diastereomeric at C-24); MS (pos. FAB) m/z 652.5 (M+1, 100%), 530.5(M+1-HOBz, 6%). Compound 2021α: ¹ H NMR (500 MHz, CD₃ OD) δ: 8.01 (d,J=8.3 Hz, 2H, benzoate-Ar H), 7.61-7.59 (m, 1H, benzoate-Ar H),7.51-7.45 (m, 2H, benzyl-Ar H), 5.12 (s, 3H, C-7 H) , 3.19-3.15 (m, 1H), 2.86 (s, 1H) , 2.70-2.60 (m, 4H, NCH₂), 2.60-2.54 (m, 2H, NCH₂),2.54-2.49 (m, 2H, NCH₂), 0.73 (s, 3H, C-18, diastereomeric at C-24); MS(pos. FAB) m/z 652.5 (M+1, 100%), 530.5 (M+1-HOBz, 10%).

Preparation of 7α-Benzoyloxy-3α-(1,5,10-triazadecyl)cholestan-24ε-sulfate (2022):

Compound 2021α (0.0214 g, 0.0328 mmol) was converted to compound 2022(0.0190 g, 79%) as previously described for the conversion of compound2016 to compound 2017. ¹ H NMR (500 MHz, CD₃ OD) δ:8.21-8.14 (m, 2H,benzoate-Ar H), 7.62-7.50 (m, 2H, benzoate-Ar H), 5.18-5.09 (m, 1H, C-7H), 4.22-4.16 (m, 1/2H, C-24 H), 4.10-4.06 (m, 1/2H, C-24 H), 3.43 (brs, 1H, C-3 H), 3.22-3.10 (m, 5H, CH₂ N), 3.09 (t, J=7.5 Hz, 2H, CH₂ N),3.04 (br s, 2H, CH₂ N), 2.99-2.96 (m, 2H, CH₂ N), 0.60 (s, 3/2H, C-18H), 0.52 (s, 3/2H, C-18 H) (Note: compound is a mixture of diastereomersat C-24).

Preparation of 3-Episqualamine (388):

Compound 2022 (0.066 g, 0.085 mmol) was dissolved in methanolic KOH (5%,5 ml) and refluxed for 7 hours. No starting material remained by TLC.Neutralization with 5% (v/v) concentrated hydrochloric acid in methanolfollowed by removal of the solvent and flash chromatography SiO₂, 12:4:1Ch₂ Cl₂ :MeOH:NH₄ OH) gave the desired product 2023 (0.0365 g, 67%). ¹ HNMR (500 MHz, CD₃ OD) δ: 4.14-4.09 (m, 1H, C-24 H) , 3.80 (s, 1H, C-7 H), 3.48 (s, 1H, C-3 H) , 3.24-3.15 (m, 4H, CH₂ N), 3.10 (t, J=7.4 Hz, 2H,CH₂ N), 3.01 (t, J=7.1 Hz, 2H, CH₂ N), 0.86 (s, 3H, C-19 H), 0.69 (s,3H, C-18 H); MS (pos. FAB) m/z 628.5 (M+1, 18%), 548.5 (M+1-SO₃, 65%),530.4 (M+1-H₂ SO₄, 100%); high resolution MS (pos. FAB) m/z 628.4713(calcd.: 628.4723).

Preparation of 3-Episqualamine Dessulfate(3α-(1,5,10-Trizadecyl)cholestan-7α,24ε-diol, 387):

Compound 2015α (0.089 g, 0.1397 mmol) was converted to compound 387(0.0372 g, 49%) as described for the conversion of compound 2015β tocompound 2016. ¹ H NMR (500 MHz, CD₃ OD) δ: 3.80 (s, 1H, C-7 H), 3.48(s, 1H, C-3 H), 3.24-3.15 (m, 4H, CH₂ N), 3.10 (t, J=7.4 Hz, 2H, CH₂ N),3.00 (t, J=7.3 Hz, 2H, CH₂ N), 0.86 (s, 3H, C-19H), 0.69 (2s, 3H, C-18H), MS (pos. FAB) m/z 548.5 (M+1, 100%); high resolution MS (pos. FAB)548.5162 (calcd.: 548.5155).

Example S

Preparation of compound 399: ##STR22##

Preparation of 3-oxo-4-cholenic acid methyl ester 3002:

A solution of 3β-hydroxy-5-cholenic acid methyl ester 3001 (24.16 g,57.11 mmol), aluminum tri-t-butoxide (56.27 g, 228.43 mmol) andisopropylmethylketone (50 ml) in dry toluene (150 ml) was stirred andheated to 120° C. (oil bath) for 6 hours. The reaction mixture was thencooled to room temperature diluted with toluene (100 ml) and acidifiedwith 2NHCl (70 ml). The organic layer was separated, and the aqueouslayer extracted with toluene (3×50 ml). The combined organic extractswere washed with water (1×50 ml), saturated NaHCO₃ (2×50 ml), water(1×50 ml), brine (1×50 m), dried (MgSO₄), filtered and evaporated invacuo to get the crude product. Flash chromatography of the crudeproduct using toluene followed by a gradient of ethyl acetate/hexane (5,10, 20 and 40%) solvent systems gave a pure white solid,3-oxo-4-cholenic acid methyl ester 3002 (13.43 g, 60%). ¹ H NMR (400MHz, CDCl) δ: 0.71 (3H, s, 18-CH₃), 0.90 (3H, d, 21-CH₃), 1.17 (3H, S,19-CH₃), 3.66 (3H, s, CO₂ CH₃) and 5.71 (1H, s, 4-H).

Preparation of 3-oxo-5α-cholanic acid methyl ester 3003

Into a solution of 3-oxo-4-cholenic acid methyl ester 3002 (13.0 g,23.68 mmol) in dry ether (50 ml) was added distilled liquid ammonia (70ml) at -78° C. Lithium (1.0 g, 144.1 mmol) was added in small portionsuntil a blue coloration persisted for 10 minutes, after which thesolution was quenched with solid NH₄ Cl (50 g). Ammonia was evaporated,and the resulting residue was partitioned between water (100 ml) andether (150 ml). The aqueous solution was extracted further with ether(3×50 ml). The combined extracts were washed with brine (1×70 ml), dried(MgSO₄), filtered and concentrated in vacuo to get the crude product.Flash chromatography of the crude product in silica gel using ethylacetate:hexane (2:8) gave pure 3-oxo-5α-cholanic acid methyl ester 3003(3.85 g, 29%). ¹ H NMR (400 MHz, CDCl₃) δ: 0.69 (3H, s, 18-CH₃, 0.91(3H, d, 21-CH₃), 1.02 (3H, s, 19-CH₃) and 3.66 (3H, s, CO₂ CH₃).

Preparation ofN-(3'-Aminipropyl)-N,N'-(di-tert-butoxycarbonyl)-1,4-diaminobutane 301:

(a) To a solution of 1,4-diaminobutane (8.6 g, 97.6 mmol) in methanol(3.0 ml) was added a solution of acrylonitrile (6.2 g, 116.8 mmol) inmethanol (3.0 ml) at 0° C., and the mixture was stirred for 12 hours.Evaporation of the solvent in vacuo affordedN-(2'-cyanoethyl)-1,4-diaminobutane as a colorless oil (11.0 g, 80%). ¹H NMR (400 MHz, CDC₃) δ: 1.45 (4H, br, --CH₂ CH₂ --), 2.46 (2H, t), 2.58(2H, t), 2.62 (2H, t) and 2.84 (2H, t).

(b) To a solution of N-(2'-cyanoethyl)-1,4-diaminobutane (5.6 g, 40mmol) in dichloromethane (140 ml) was added dropwise a solution ofdi-t-butyldicarbonate (19.2 g, 88 mmol) in dichloromethane (20 ml) atroom temperature, and the mixture was stirred for 12 hours. The organicsolvent was removed in vacuo and the residual oil was dissolved in ethylacetate (150 ml), and washed with saturated NaHCO₃ (2×75 ml), water(2×75 ml), brine (75 ml), dried (MgSO₄), filtered and evaporated to getthe crude viscous oil. The crude product was purified by flashchromatography in silica gel to give pure(N-(2'cyanoethyl)-N,N'-(di-t-butoxycarbonyl)-1,4-diaminobutane as acolorless viscous oil (8.4 g, 75%). ¹ H NMR (400 MHz, CDCl₃) δ: 1.44(9H, s, t-Boc), 1.46 (9H, merged s, t-Boc), 2.60 (2H, m), 3.15 (2H, m),3.28 (2H, t) and 3.45 (2H, t); CIMS (m/e): 342 (M+1, 62.7%), 239 (100%),186 (83.1%)

(c) To a suspension of lithium aluminum hydride (1.8 g, 48.9 mmol) indry ether (300 ml) was added a solution ofN-(2'-cyanoethyl)N,N'-(di-t-butoxycarbonyl)-1,4-diaminobutane (4.8 g,13.8 mmol) in dry ether (150 ml) dropwise at 0° C., and the mixture wasstirred for 30 minutes. The excess lithium aluminum hydride was quenchedwith 1N NaOH at 0° C. and the resulting white suspension wads filteredthrough Celite and washed with ether, and the ether extract was washedwith brine, dried (MgSO₄), filtered and evaporated in vacuo to get acrude oil. The crude product was purified by flash chromatography insilica gel to give pureN-(3'-aminopropyl)-N,N'-(di-t-butoxycarbonyl)-1,4-diaminobutane 301 (3.3g. 68%) as a colorless oil. ¹ H NMR (400 MHz, CDCl₃) δ: 1.44 (18H, s,2(t-Boc)), 2.68 (2H, t), 3.05-3.25 (6H, br), and 4.65 (1H, br); CIMS(m/e): 346 (M⁺ +1, 100%), 290 (3.1%), 246 (32.2%).

Preparation of 3β-N-1-{N3-(4-Aminobutyl)!-1,3-diaminopropane}-24-hydroxy-5α-cholanetrihydrochloride 3005:

To a solution of 3-oxo-5α-cholanic acid methyl ester 3003 (3.0 g, 7.73mmol) andN-(3'-aminopropyl)-N,N'-(di-t-butoxycarbonyl)-1,4-diaminobutane 301(4.01 g, 11.60 mmol) in methanol (150 ml) was added 3 Å molecular sieves(10 g) and NaCNBH₃ (0.73 g, 11.61 mmol). The reaction mixture wasstirred at room temperature for 16 hours. After filtering throughCelite, methanol was removed in vacuo. The residue was dissolved inmethanol (50 ml) and then methanol pre-saturated with HCl gas (15 ml)was added. The reaction mixture was stirred at room temperature for 6hours. After removing methanol in vacuo, the crude product was dissolvedin tetrahydrofuran (100 ml) and then lithium aluminum hydride (1.50 g,39.52 mmol) was added in one portion. The reaction mixture was gentlyrefluxed for 8 hours. The reaction mixture was cooled to 0° C. (icebath), then a solution of 2N NaOH was added dropwise until white solidgranulates were formed. Tetrahydrofuran was decanted and the residuefurther extracted with toluene (3×50 ml), and the combined organicextracts were dried (K₂ CO₃), filtered and evaporated in vacuo to getthe residue. The residue was dissolved in dry methanol (50 ml) and thenmethanol presaturated with HCl gas (20 ml) was added. After one hour,excess methanol was removed in vacuo to get white solid. The crudeproduct was purified by flash chromatography in silica gel usingchloroform:methanol:isopropylamine (15:1:1) to get pure 3β-N-1-{N3(4-aminobutyl)!-1,3-diaminopropane}-24-hydroxy-5α-cholanetrihydrochloride 3005 (1.10 g, 24%). ¹ H NMR (400 MHz, CD₃ OD) δ: 0.71(3H, s, 18 -CH₃), 0.89 (3H, s, 19-CH₃), 096 (3H, d, 21-CH₃), 2.90-3.40(9H, m) and 3.51 (2H, br t, CH₂ O); MS-FAB (positive): 490 (M⁺ +1,100%), 419 (8%) and 360 (7.5%)

Preparation of 3β-N-1-{N3-(4-Trifluoroacetyl)aminobutyl)!-1,3-di(trifluoroacetyl)diaminopropane}-24-hydroxy-5α-cholane3006:

To a solution of 3β-N-1-{N3-(4-aminobutyl)!-1,3-diaminopropane}-24-hydroxy-5α-cholanetrihydrochloride 3005 (0.95 g, 1.58 mmol) in dry methanol (20 ml) wasadded dry triethylamine (2.29 ml, 15.8 mmol) followed by ethyltrifluoroacetate (2.80 ml, 23.53 mmol) at room temperature, and themixture was stirred for 20 hours. After removal of excess methanol andlow boiling organic reagents in vacuo produced a white residue. Theresidue was dissolved in ethyl acetate (50 ml) and then washed with 2NHCl (3×20 ml), water (2×20 ml), saturated NaHCO₃ (3×20 ml) and brine(1×20 ml), dried (MgSO₄), filtered and evaporated in vacuo to get analmost pure white solid, 3β-N-1-{N3-(4-trifluoroacetyl)aminobutyl)!-1,3-di(trifluoroacetyl)diaminopropane}-24-hydroxy-5α-cholane 3006 (0.77 g,73%). ¹ HNMR (400 MHz, CDCl₃) δ: 0.71 (3H, s, 18 -CH₃), 0.89 (3H, s,19-CH₃), 0.96 (3H, d, 21-CH₃), 3.01-3.57 (11H, m, 4xCH₂ N+1xCHN+CH₂ O).

Preparation of 3β-N-1-{N 3-4-Trifluoroacetyl)aminobutyl)!-1,3-di(trifluoroacetyl)diaminopropane}-24-hydroxy-5α-cholane24-pyridinium sulfate 3007:

To a solution of compound 3006 (0.70 g, 1.05 mmol) in dry pyridine (20ml) was added sulfur trioxide pyridine complex (0.75 g, 4.71 mmol) atroom temperature, and the mixture was stirred for 6 hours. The excesspyridine was removed in vacuo to get solid residue, from which thesulfated compound was extracted with chloroform (5×20 ml). Removal ofchloroform gave white solid, 3β-N-1-{N3-(4-trifluoro-acetyl)aminobutyl)!-1,3-di(trifluoroacetyl)diaminopropane}-24-hydroxy-5α-cholane24-pyridinium sulfate 3007 along with excess reagent (1.0 g). The crudeproduct was used in the next step without further purification.

Preparation of 3β-N-1-{N3-(4-Aminobutyl)!-1,3-diaminopropane}24-hydroxy-5α-cholane 24-potassiumsulfate (399):

To a solution of crude compound 3007 (1.0 g) in methanol (25 ml) wasadded a solution of potassium carbonate in water (10 ml) at roomtemperature, and the mixture was stirred overnight. After 18 hours, theexcess methanol and water were removed in vacuo to get the residue. Theresidue was extracted with methanol (3×30 ml). The combined methanolextracts were concentrated in vacuo to get crude product. Flashchromatography of the crude product in silica gel usingdichloromethane:methanol:ammonium hydroxide (7:2:1) (dried over MgSO₄before use) gave white solid, 3β-N-1-{N3-(4-aminobutyl)!-1,3-diaminopropane}-24-hydroxy-5α-cholane 24-potassiumsulfate or compound 399 (0.22 g, 35% based on compound 3006). ¹ H NMR(400 MHz, CD₃ OD) δ: 0.74 (3H, s, 18 -CH₃), 0.92 (3H, s, 19-CH₃), 1.0(3H, d, 21-CH₃), 2.95-3.24 (9H, m) and 4.00 (2H, br t, CH₂ OSO₃); MS-FAB(positive) (m/e): 570 (M⁺ +2, 85%), 490 (44%), 430 (15%), 402 (16%);MS-FAB (negative) (m/e): 568 (M⁺, 3.7%), 495 (10%), 452 (7%), 438 (17%),423 (14%).

Example T

Preparation of compound 1436:

This compound can be readily prepared from squalamine through thecoupling of β-alanine aldehyde, followed by reduction, as shown in thefollowing scheme: ##STR23##

The above approach permits the ready conversion of squalamine, presentin large amounts in shark liver, to compound 1436, present in about 5%the quantity of squalamine.

Additional aminosterol compounds such as those shown in Tables I and IIherein can be prepared in manners analogous to those given above.

Therapeutic Activities and Utilities

Aminosterol compounds such as squalamine have been discovered to beeffective inhibitors of NHE. In seeking to elucidate the antimicrobialmechanism of action for squalamine, squalamine has been found toadvantageously inhibit a specific NHE isoform--the compound inhibitsNHE3, but not NHE1. In addition, squalamine has been determined toinhibit the exchanger through a special mechanism. The special andadvantageous effects and utilities of squalamine and other aminosterolsare further evident from the results of the experimental tests discussedbelow.

Specific Inhibition of NHE3:

To determine the specificity of squalamine's inhibition of NHEs,squalamine was assayed against a cell line expressing either human NHE1or human NHE3 following procedures outlined in Tse et al., J. Biol.Chem. 268, 1993, 11917-11924. Internal pH was measured either followingacid loading or in the absence of an acid-loading challenge, with theresults shown in FIGS. 1A and 1B.

Specifically, PS120 fibroblasts transfected with rabbit NHE3 were grownin supplemented Dulbecco's-modified Eagle's medium as described byLevine et al., J. Biol. Chem. 268, 1993, 25527-25535. Transfected cellsgrown on glass coverslips were then assayed for internal pH changesfollowing treatment with 5 μg/ml squalamine using the fluorescent dyeBCECF-AM (2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethylester) as a pH indicator as described by Levine et al. For cellsacid-preloaded by exposure to 40 mM NH₄ Cl, the rate of pH recovery as afunction of restored extracellular sodium ion concentration wasmonitored, with the results being shown in FIG. 1A. For cells notacid-preloaded, the actual internal pH value was monitored as a functionof time following addition of squalamine, with the results depicted inFIG. 1B.

As seen in FIGS. 1A and 1B, squalamine inhibited NHE3 with respect toproton concentration at both K_(m) and V_(max) levels. In contrast,existing agents such as amiloride affected only V_(max).

Thus, the aminosterol squalamine not only reduces the absolute number ofprotons that can be secreted by the cell (the V_(max) effect), but alsoforces the cell to fall to a lower pH in the presence of this inhibitor(the K_(m) effect). As a consequence, the sodium/proton exchanger ismore profoundly inactivated by squalamine than by amiloride.

In contrast to its effects on NHE3 shown in FIGS. 1A and 1B, squalamineexhibited no inhibitory activity against human NHE1 or rabbit NHE1 asshown in FIGS. 2A and 2B. PS120 fibroblasts transfected with rabbit orhuman NHEl were grown as described above. Transfected cells expressingrabbit NHE1 (FIG. 2A) or human NHE1 (FIG. 2B) grown on glass coverslipswere then assayed for internal pH changes following treatment with 5μg/ml squalamine using the fluorescent dye BCECF-AM with cellsacid-preloaded by exposure to 40 mM NH₄ Cl. The rate of pH recovery as afunction of restored extracellular sodium ion concentration wasmonitored.

In addition, as demonstrated by FIG. 1B, the resting pH of these cellswas also inhibited. Thus, squalamine's effect on proton exchange causesthe cell to drop to a lower pH in its presence before activation of thepump occurs.

Through these studies, squalamine has been discovered to be a distinctinhibitor with specificity for NHE3 over NHE1. Moreover, squalamine hasbeen identified as an inhibitor that causes a cell to drop to a lower pHbefore the pump is activated. The results shown in FIGS. 1A, 1B, 2A and2B demonstrate that squalamine exhibits a unique NHE specificity.

The Expression of NHE3:

Such a specific effect for NHE3 is important for several reasons. NHE3,being present on apical surfaces of a limited number of cell types, ismore specialized than NHE1. A cell of particular interesttherapeutically is the endothelial cell.

Using PCR, the expression of this antiporter in both human microvascularand human pulmonary artery endothelial cells has now been demonstrated.Total RNA was isolated from primary human pulmonary artery endothelialcells (HPAEC), human melanoma cell line wm1617, and human microvascularendothelial cells (HMVEC) by a modification of the method of Chomczynskiet al., Anal. Biochem. 162, 1987, 156, and then reverse-transcribed withMMLV reverse transcriptase using a first strand cDNA synthesis kit(Clontech Laboratories, Palo Alto, Calif.). Human small-intestine totalRNA obtained from Clontech was also reverse-transcribed in the samefashion.

Approximately 80 ng of the cDNA product were then amplified in a 50-μlreaction mixture using reagents from the AmpliTaq DNA Polymerase kit(Perkin Elmer, Norwalk, Conn.) and containing 400 ng each of twooligonucleotides specific for human NHE3 (B13: 5'CATCTGGACCTGGAACACG-3';B14: 5'-CGTAGCTGATGGCATCCTTC-3') using one thermal cycle of 5',94° C.,and then 38 cycles of 50", 94° C., 1',57° C.,2',71° C., and finally10',72° C. before cooling to 4° C. Twenty μl of this sample was analyzedon a 1.7% agarose gel. The expected NHE3 PCR band of about 550 bp wasseen in most instances, as indicated in Table 2 below.

One μl of each PCR reaction was then further analyzed by nested PCR in a50-μl reaction mix using two internal primers (B15:5'-CTGGTCTTCATCTCCGTGTAC-3'; B16: 5'-AGCTCGTGGAA-GACATTCAGG) with a5',94° C. program of thermal cycling, then 35 cycles of 50",94° C.,1',58° C., 2',71° C., and finally 10',72° C. before cooling to 4° C.About 20 μl of this reaction was analyzed on another 1.7% agarose gel.The expected NHE3 PCR band of about 490 bp was seen in all cases asnoted in the table below. DNA sequencing of the HPAEC and HMVEC nestedPCR bands from both ends confirmed they had the expected human NHE3sequences.

                  TABLE 2                                                         ______________________________________                                        EXPRESSION OF HUMAN NHE3 IN HUMAN                                             ENDOTHELIAL CELL LINES                                                                  Visible Detection of Human NHE3 by PCR                              Total RNA Source                                                                          1 PCR Round   2 Nested PCR Rounds                                 ______________________________________                                        Small Intestine                                                                           -             +                                                   Human Melanoma                                                                            +             +                                                   HPAEC       +             +                                                   HMVEC       +/- (multiple bands)                                                                        +                                                   ______________________________________                                    

Thus, a variety of endothelial cell growth/shape related events areinhibited by squalamine and functionally related compounds. Theexperimental tests discussed below were conducted to assess thisaminosterol's effects.

Growth Inhibition of Endothelial Cells, Fibroblasts and Epithelial CellsIn Vitro

When non-transformed human cells are grown in the presence of increasingconcentrations of squalamine, endothelial cells exhibit a particularsensitivity to squalamine, as shown by the following experiment. Bovinepulmonary endothelial cells, human epithelial cell line MCF 10A, andhuman foreskin fibroblasts were incubated in the presence of 12different membrane-active agents, including peptides and squalamine.

Specifically, bovine pulmonary endothelial cells, human epithelial cellline MCF 10A, and human foreskin fibroblasts were incubated in thepresence of the following twelve membrane-active agents: (1) RGDKIAGKIA!₃ -NH₂ ; (2) d- KKLLKKL!₂ -NH₂ ; (3) squalamine; (4)SWLSKTAKKLENSAKKRISEGIAIAIQGGPR; (5) FLGGLIKIVPAMICAVTKKC; (6) Magainin2; (7) PGLA; (8) GFASFLGKALKAALKIGANLLGGTPQQ; (9) PR-39; (10) 1-KKLLKKL!₂ -NH₂ (11) Cecropin B; and (12) KIAGKIA!₃ -NH₂. Cell growth wasmeasured by absorbance at 600 nm. Results are shown in FIGS. 3A-3C.

As evident from FIG. 3A, squalamine inhibited the growth of bovinepulmonary artery endothelial cells (BPE) at 1 μg/ml. In contrast, at 10μg/ml it exerted no effect on the growth of either epithelial (FIG. 3B)or fibroblast (FIG. 3C) lines. However, peptides that inhibited thegrowth of epithelial cells exhibited no effect on BPE. Thus, endothelialcells are more sensitive to squalamine than are either fibroblasts orepithelial cells.

Inhibition of Endothelial Cell Cord Formation In Vitro

Endothelial cells have the capacity in vitro to form tubular aggregatesresembling capillaries in various early stages of formation. Thisconversion occurs under relatively specific conditions, in whichessential growth factors along with an effective substratum areprovided. It has been shown that both the interaction of growth factorswith the endothelial cell and its attachment to a substratum activatethe NHE. The activation of this exchanger is believed to be required forsubsequent morphologic transformation of the endothelial cell into amulticellular tubular structure.

To assess the effect of compounds on the cord-like structures formed byhuman microvascular cells when plated in the presence of VEGF (VascularEndothelial Growth Factor) and basic fibroblast growth factor on acollagen matrix, a standard cord formation assay was used. The resultsare shown in the table below.

                  TABLE 3                                                         ______________________________________                                        EFFECT OF VARIOUS AMINOSTEROLS ON ENDOTHELIAL                                 CORD FORMATION                                                                           μg/ml                                                                      0.01   0.1      1.0      10.0                                      ______________________________________                                        Fumagillin            -        +/-    +                                       Squalamine   -        +        +      +                                       Compound 319 -        +        +      +                                       Compound 353          +        +      +                                       Compound 410          -        +      +*                                      Compound 411          -        -      +                                       Compound 412          -        -      +                                       Compound 413          -        -      +                                       Compound 415          -        -      +/T                                     Compound 371                   T      T                                       Compound 432                   -      -                                       Compound 449                   -      +/-                                     Compound 467                   -      -                                       ______________________________________                                         Notes:                                                                        + = Inhibition of angiogenesis;                                               - = No inhibition of angiogenesis;                                            T = Toxic;                                                                    *= cell rounding @ 10 μg/ml.                                          

As shown in Table 3, squalamine inhibits cord formation at about 0.1μg/ml, compared with fumagillin, which exhibits comparable activity at10 μg/ml. At these concentrations, squalamine does not appear toprofoundly affect cell viability or proliferation. This property invitro roughly correlates with anti-angiogenic activity in more complexin vivo models (see Goto et al., Lab Investigation 69, 1993, 508-518).

LPS-Induced Neutrophil Adherence to Human Umbilical Venous EndothelialCells

When endothelial cells are exposed to certain stimuli, includinglipopolysaccharide (LPS) and certain cytokines, specific adhesionmolecules are induced on the plasma membrane that enhance the binding ofleukocytes. These leukocyte-endothelial cell interactions are believedto be necessary to localize leukocytes to sites of bacterial invasionand to facilitate extravasation of the leukocytes from the capillaryinto the surrounding tissue space. Leukocyte-adhesion molecules includethe Selectins and ICAM-1.

To determine if squalamine inhibited this particular endothelial cellfunction, standard adhesion assays were performed as outlined in Gambleet al., J. Imm. Methods 109, 1988, 175-184. The expression of cellsurface ligands in an endothelial-based system has been shown to effectadherence to granulocytes with a system using human umbilical venousendothelial cells, purified neutrophils, and inducers of cell surfaceligands such as LPS (100 ng/ml) and TNF-α (40 ng/ml). In theseexperiments, approximately 2×10⁵ human umbilical venous cells (passage2-6) were plated per well. The cells were grown in serum-free mediaovernight. For induction, either TNF-α (40 ng/ml) was added toendothelial cells for 6 hours prior to adding neutrophils or LPS (100ng/ml) was added for 4-6 hours. It was found that the LPS response wasincreased by adding 1% FBS to the wells to provide a source ofLPS-binding protein. After activation of the endothelial cells,approximately 50×10⁶ neutrophils were added per well. The plates weregently rocked for 30 minutes at room temperature, followed by removal ofthe media and washing in serum-free media three times and thenphotographing of each well. Experiments to test the effects ofsqualamine were performed by adding squalamine at 10 μg, 1.0 μg, or 0.1μg at the time of adding LPS or TNF-α. A second repeat dose ofsqualamine was added at the time of adding neutrophils. A monoclonal Abto ICAM-1 was a positive control.

Using three different subjects, there was no inhibition of squalamine onneutrophil adherence using activated human endothelial cells. There wasapproximately 50% inhibition of adherence when adding 40 μg/ml of amonoclonal Ab to ICAM-1 prior to adding neutrophils.

These results indicate that inhibition of the endothelial NHE bysqualamine affects both growth and capillary formation in vitro, butdoes not inhibit all signal transduction pathways in this cell. Thus,certain "housekeeping" functions, such as the capacity of theendothelial cell to attract leukocytes to the site of an infection,should not be impaired by squalamine. This demonstrates that squalaminecan be used to inhibit angiogenesis but will not otherwise disruptcertain important endothelial cell functions, such as leukocyterecruitment to sites of infection or inflammation.

Anti-proliferative Activity

The Chorioallantoic Membrane Model

Using the classical chorioallantoic membrane model, it has been foundthat squalamine is an inhibitor of capillary growth. The growingcapillaries within the chorioallantoic membrane model (CAM model) havebeen used as a system in which to evaluate the effect of agents on theirpotential to inhibit new vessel growth. Neovascularization occurs mostaggressively over the first week of embryonic development. Thereaftercapillary growth is characterized by principally "elongation" ratherthan "de novo" formation.

In the standard assay, agents are applied locally to a region of theembryo over which neovascularization will occur. Agents are assessed bytheir ability to inhibit this process, as evaluated by visualexamination about 7 days after application. Agents which disruptvascular growth during the period of de novo capillary formation, but donot interfere with subsequent capillary growth, are generally regardedas "specific" inhibitors of neovascularization, as distinguished fromless specific toxic substances. The assay utilized is described indetail in Auerbach et al., Pharm. Ther. 51, 1991, 1-11. Results aretabulated below.

                  TABLE 4                                                         ______________________________________                                        INHIBITION OF CAPILLARY GROWTH IN CAM MODEL                                   ______________________________________                                        3-Day     Squalamine                                                                              Percentage positive                                       Embryo:   Applied(μg)                                                                          Assay 1   Assay 2                                                                             Mean                                      ______________________________________                                                  0.65      28                                                                  1.25      18        18    18                                                  2.5       35        18    27                                                  5.0       91        57    74                                                  20         52*       58*  55                                                  40         50*       13*  32                                        ______________________________________                                         Note:                                                                         *= Some vascular irritation noted.                                       

    13-Day:     Squalamine                                                        Embryo:     Applied (μg)                                                                         Percentage positive                                     ______________________________________                                                    5.0       0/26                                                    ______________________________________                                    

As seen from Table 4, applying as little as 0.65 μg squalamine to a3-day CAM resulted in inhibition of CAM vessel neovascularization. Incontrast, applying ten times that amount of squalamine onto a 13-day oldchick exerted no inhibitory effect.

Thus, in a classical angiogenesis assay, squalamine exhibited potent butspecific inhibitory activity, equal in potency to the most activecompounds described to date in the literature. The effect is compatiblewith suppression of neovascularization rather than toxic inhibition ofcapillary growth.

The Vitelline Capillaries of 3-5 Day Chick Embryo Model

In the course of evaluating squalamine in the "classical" chickchorioallantoic membrane model, it was noted that this steroid exerted adramatic and rapid effect on capillary vessel integrity in the three- tofive-day old chick embryo. Using the chick embryo vitelline capillariesassay, compounds were tested for their ability to induce capillaryregression. Each compound was applied in 0.1 ml of 15% Ficol 400 and PBSonto the embryo, and vascular regression was assessed after 60 minutes.

Squalamine was found to disrupt vitelline capillaries in 3- to 5-daychick embryos. The 3-day chick embryo consists of an embryonic disc fromwhich numerous vessels emerge and return, forming a "FIG. 8"-shapedstructure--the embryo in the center with vascular loops extendingoutward over both poles. Application of squalamine onto the embryonicstructure (0.1 ml in 15% Ficol in PBS) resulted in progressive "beadingup" of the vitelline vessels, with the finest capillaries being thefirst to exhibit these changes. Following a lag period of around 15minutes, the constriction of continuity between capillary and secondaryvessels, generally on the "venous" side, was observed. Continuedpulsatile blood flow progressed, resulting in a "swelling" of the blindtube, followed by a pinching off of the remaining connection andformation of an enclosed vascular sac resembling a "blood island." Thisprocess progressed until only the largest vessels remained intact. Theembryonic heart continued to beat vigorously. No hemorrhage was seen,reflecting the integrity of the capillary structure. In addition, noobvious disruption of circulating red cells was observedmicroscopically, demonstrating the absence of hemolysis.

Utilizing this assay, which appears to demonstrate what is commonlycalled capillary "regression," a minimum concentration of squalaminerequired to observe an effect in 60 minutes can be determined. Resultsare summarized in the table below.

                  TABLE 5                                                         ______________________________________                                        EFFECTS OF VARIOUS AMINOSTEROLS IN CHICK EMBRYO                               VITELLINE CAPILLARY REGRESSION ASSAY                                                     Amount of Compound Applied (μg)                                 Compound     10     1       0.1   0.01   0.001                                ______________________________________                                        Compound 1436                                                                              +      +       +     +      +/-                                  Compound 319 +      +       +     +      +/-                                  squalamine   +      +       +     +      0                                    Compound 415        +       +     +      0                                    Compound 410        +       +/-   +/-    0                                    Compound 412        +       0     0      0                                    Compound 411        +/-     0     0      0                                    Compound 382 +      +       0     0      0                                    Compound 1364                                                                              +      +       0     0      0                                    Compound 371 +      +/-     0     0      0                                    Compound 396        +       0     0      0                                    Compound 353        +/-                                                       Compound 413        0                                                         Compound 414        0                                                         Compound 381        0                                                         Compound 303        0                                                         Compound 318        0                                                         Compound 409        0                                                         Compound 1360       0                                                         Vehicle      0      0       0     0      0                                    ______________________________________                                         Notes:                                                                        + = Vascular reactivity;                                                      0 = No vascular reactivity;                                                   +/- = Equivocal reactivity;                                                   Vehicle = 15% (w/w) Ficol in phosphatebuffered saline.                   

As apparent from Table 5, 0.1-0.01 μg of squalamine in 0.1 ml medium caninduce changes. Compounds having various ranges of activities werefound, with squalamine, compound 319 and compound 415 being especiallyactive. This experiment demonstrates that the steroids tested candramatically restructure capillaries over a time interval amounting toseveral minutes. The results reflect that squalamine exerts this effectthrough inhibition of NHE.

Tadpole Assay

A newly developed assay employing tadpoles, preferably Xenopus laevisStages 59-60, were employed to study the effect of a compound bymonitoring capillary occlusion in the tadpole's tail. Animals at thesestages were used because they represent the period of transition throughmetamorphosis at which time the animals possess both embryonic and adultstage tissues. The compounds of the invention affect the shape,viability and integrity of the embryonic tissues while not affecting theadult tissues, providing a powerful, highly specific screen. Forexample, substances that destroy all of the animal's epithelium, bothadult and embryonic, could be regarded as toxic. Substances that destroyonly the embryonic tissues exhibit a very unique specificity.

In this assay, tadpoles are introduced into Petri dishes containing asolution of the test compound in distilled water, preferably about 100ml. The preferred concentration of the test compound is from about 1μg/ml to about 10 μg/ml. The volume of liquid is sufficient for theanimal to swim freely and drink from the solution. Thus, the effectobserved results from oral absorption and subsequent systemicdistribution of the agent. If the volume of liquid is not sufficient topermit oral intake, the effects that are observed would result fromabsorption through the surface epithelium. Thus, this simple assay canidentify if an compound has characteristics of oral availability.

In another embodiment of this assay, a solution of a compound in watercan be injected directly into the abdomen of the animal using standardtechniques. Concentrations of the compound from about 0.05 mg/ml toabout 0.5 mg/ml in about 0.05 ml of water are preferred.

After an amount of time, typically about 60 minutes, the occlusion ofblood flow through capillaries in the tadpole's tail are observed underan inverted microscope at a magnification of roughly 100X.

When the tadpoles were introduced into distilled water containingsqualamine at 10 μg/ml, it was observed that blood flow through thecapillaries of the tail shut down. The process occurred from the caudalto cranial direction. Blood flow within the most distal vessels stoppedinitially, followed by the larger vessels. During this period, it wasobserved that the cardiovascular system was otherwise robust, asevidenced by a continued heartbeat, pulsatile expansion of the greatvessels, and, most curiously, unaltered blood flow through the finecapillaries of the hands and feet. Thus, selective cessation of bloodflow was seen in localized regions. If the animals are maintained insqualamine for several days, enhanced regression of the most distalaspects of the tail, as well as the peripheral aspects of the tail finare observed, corresponding to regions of the animal perfused by theoccluded vasculature.

This effect apparently results from selective change in the restingdiameter of the capillaries of the tail. Inhibition of the endothelialcell NHE evidently leads to a change in shape of the cell making up thecapillary, resulting in diminished flow. The continued functioning ofcapillary beds in the "adult" portions of the tadpole (the limbs)indicates that squalamine is selective for certain capillaries. From theresults of the tadpole tail capillary occlusion assay, compound 319,squalamine and compound 1436 were found to induce a common vascularocclusive effect.

Suppression of Melanoma Growth

Suppression of Growth of Melanomas in Mice by Oral and Parenteral Routesof Administration

The growth of B16 melanoma in C57B mice is dependent uponneovascularization. Hence, this is a recognized model for evaluating theimpact of inhibitors of angiogenesis on the growth of cancer.

Using the growth of B16 melanoma cells in C57B mice, a recognized modelfor the evaluation of inhibitors of angiogenesis on the growth ofcancers, the effects of subcutaneous, intraperitoneal and oraladministration of squalamine were evaluated. An inoculum of B16 melanomacells was implanted subcutaneously on the dorsum of the C57B mouse,which resulted in the progressive growth of melanoma lesions over 30-40days as shown in FIGS. 4A, 4B, and 4C.

In this model, there was observed little evidence of metastasis with orwithout treatment with chemotherapeutic agents. When animals weretreated with squalamine either subcutaneously (FIGS. 4A and 4A-1),intraperitoneally (FIGS. 4B and 4B-1), or orally (FIGS. 4C and 4C-1), adose-dependent suppression of tumor volume was observed. Measurement ofboth body weight and hematologic parameters demonstrated no significantdepression. Since squalamine itself shows minimal cytostatic activityagainst B16 in culture, except at very high concentrations, thisresponse of the tumor was interpreted to be secondary to interferencewith capillary development.

Suppression of Growth of Human Melanoma in Immunocompromised Mice

As apparent from FIG. 5, melanoma 120SLu develops aggressively in RAG-1mice after implantation. Squalamine has been found to suppress thegrowth of melanoma 1205Lu in RAG-1 mice in a dose-dependent fashion.

Squalamine was administered after tumors had reached about 0.1 ml, andclear suppression of tumor growth in a dose-dependent fashion was foundas evidenced by FIG. 5. After cessation of treatment, tumor growthcontinued at a rate similar to untreated controls, suggesting that theimpact of squalamine in this setting is reversible.

Suppression of Tumor-Induced Corneal Neovascularization in Rabbits

The implantation of VX2 carcinoma into the rabbit cornea results in theinduction of new blood vessels within several days (Tamargo et al.,Cancer Research 51, 1991, 672-675). It is believed that this carcinomasecretes growth factors that stimulate new blood-vessel growth. Thus,this model is indicative in vivo evidence of therapeutic utility in thetreatment of pathological disorders of vascularization, including themetastatic spread of tumors, diabetic retinopathy, macular degeneration,and rheumatoid arthritis.

This experiment followed the published protocol--tumor was implantedadjacent to a polymer containing a concentration of the agent to beevaluated. The polymer releases the agent slowly in the immediateneighborhood of the tumor, providing sustained high local concentrationsof the agent. In this experiment, squalamine introduced into a pellet ofELVAX 40 P (DuPont, Wilmington, Del.) inhibited new blood vesselformation by about 60% at days 7 and 14, and by about 25% at day 21.

As demonstrated by the experiments described above, squalamine providesa potent inhibitor of NHE3. Squalamine therefore should provideinvaluable therapeutic intervention wherever new blood vessel formationin vivo is implicated.

Indeed, any pathological processes dependent on new blood vesselformation can be treated through inhibition of NHE3. As an agent thatinterferes with the process of neovascularization, squalamine hastherapeutic utility in the treatment of diseases or disorders dependenton continued neovascularization where interruption of neovascularizationdiminishes the intensity of the pathological process. Thus, squalaminehas utility for treating disorders including solid tumor growth andmetastasis, rheumatoid arthritis, psoriasis, diabetic retinopathy,macular degeneration, neovascular glaucoma, papilloma, retrolentalfibroplasia, and organ rejection.

Moreover, other aminosterols have shown anti-angiogenic activity.Compounds were subjected to a various assays, including the chick embryocapillary regression assay, the tadpole assay, the assay for inhibitionof endothelial cord formation, and the assay for direct inhibition ofNHE, as described above, to determine their utilities. As evident fromthe above data, correlation among the results from the chick, tadpoleand in vitro inhibition of endothelial cell cord formation assays isexcellent.

Through the application of these assays, compound 319 emerged as anattractive alternative to squalamine. In fact, it has been found to besuperior to squalamine in the following characteristics: potency as anNHE inhibitor; simpler synthetic route; specificity--i.e, no CNSeffects. Further properties of compound 319 are discussed below.

Melanoma Growth Suppression

Compound 319 has been found to exhibit activity against B16 melanoma invivo. As seen in FIG. 6, which illustrates the results from the murinemelanoma assay described above, subcutaneous administration of thecompound achieved control of B16 in C57B mice to an extent almostcomparable to squalamine (FIG. 4B).

Pharmakokinetic Clearance

Compound 319 also has a more rapid pharmacokinetic clearance thansqualamine. To assess clearance, a mouse IV PK study was performed forcompound 319 and squalamine. The compound was administered i.v. andblood samples were taken every 10 minutes. The concentration of theadministered steroid was determined by HPLC analysis. As shown in FIG.7, after i.v. administration, the compound was cleared from the bloodstream of the mouse with a half-life of about 14 minutes. In comparison,squalamine was cleared with about a 35-minute half-life, as reflected byFIG. 8.

It should be possible to achieve further reductions in clearance in vivothrough derivatives of compound 319. It is frequently of value to extendthe lifetime of an agent in the bloodstream, to achieve higher bloodlevels with a given dose of drug and to reduce the frequency ofadministration. Polyamines are readily metabolized by a variety ofoxidases, which degrade the free terminal amino group of the polyaminemoiety. See Seller et al., Prog. Drug. Res. 43, 1994, 88-126. Alkylationof the primary amine generally retards this metabolic pathway. Seller etal., id. Thus, through simple alkylation of the primary amine oncompound 319 or on any of the steroids bearing a metabolizablepolyamine, straightforward modifications of this type would be expectedto extend biological lifetime.

Xenopus Tadpole Assay

The tadpole assay described above provides an advantageous way todetermine the pharmacological targets of each steroid when introducedinto a mammal, and to determine pharmacological categories into whichsynthetic compounds belong. In the assay, each of the steroids wasdissolved in 100 ml of distilled water at a concentration of 10 μg/ml.Stage 59-60 Xenopus tadpoles were introduced and evaluated by lightmicroscopy and gross observation 1 hour later.

The steroids tested were observed as producing different and distinctivepharmacological responses in this animal:

Compound 1256 (Squalamine): Vascular occlusion in fine capillaries oftail. No effect on vascular flow through hands or feet. Inactivity anddeath occurred within 2 hours.

FX1: Increased passage of fecal material within 1 hour. By 12 hours,solution contained considerable fecal debris. Circulatory system ofanimal appeared hyperemic, suggestive of hematopathic stimulation.

Compound 1360: Swelling and lysis of certain erythrocytes occurred,resulting in accumulation of nuclei within certain small vessels of thetail. Subsequent tissue breakdown occurred in areas of tail surroundingthese nuclear plugs.

Compound 1361: Similar to compound 1360.

Compound 1436: Gradual reduction in overall activity. Heart beatremained strong, suggesting nervous system depression. Melanocytes overhead and tail began to swell, first exhibiting visibly distinct nuclei,followed by rupture into fragments. Animal died by about 2 hours.

Compound 1437: Epithelium covering the embryonic portions of the animal,such as the tail and antennae, began to slough off in sheets. Sheets ofcells remain intact initially, but gradually detach from one another.Trypan Blue staining demonstrates that cell death occurred. Animal wasotherwise active, with little tissue breakdown noted.

FX 3: Muscular bundle within the tail began to leak myoglobin.Striations of the skeletal muscle grew less distinct. Segments of musclebegan to separate.

Inhibition of Mitogen-Stimulated Growth of Human T-cells

Specific assays were used to identify steroids with a particularbiological activity, such as an assay for inhibition ofmitogen-stimulated growth of human T-cells. Mitogen-induced cellproliferation has been reported to be dependent on the activation of theNHE. Thus, to determine which steroids act on particular cells, one needonly determine which steroids inhibit mitogen- (or growthfactor)-activated cellular proliferation.

The T lymphocyte is the lymphoid cell which serves as the host of HIVinfection. A steroid that inhibits transformation of human lymphocytesis, in principle, acting on an NHE probably activated during HIVinfection. Indeed, since GP120 activates hippocampal cell NHE uponbinding to its cellular receptor (Benos et al., J. Biol. Chem. 269,1954, 13811-13816), the assumption that a similar event follows earlyviral interaction with the lymphocyte is reasonable. This formed thebasis of the next assay.

Human heparinized blood, freshly collected, was introduced into tissueculture flasks containing 10 μg/ml phytohaemagglutinin (PHA) in RPMImedium with 10% FCS. Various purified steroids were introducedsubsequently at concentrations of 1, 5, and 10 μg/ml. Cultures wereincubated for 72 hours, after which time colcemid was added to 1 μg/ml.Cultures were maintained for an additional 2 hours, and cells collected.Mitotic figures were estimated using standard cytochemical techniques,following Giemsa staining. Results are tabulated below.

                  TABLE 6                                                         ______________________________________                                        INHIBITION OF PHA-STIMULATED HUMAN LYMPHOCYTES                                         (% control)                                                          Compound   1 μg/ml  5 μg/ml                                                                            10 μg/ml                                    ______________________________________                                        1256       3           8       10                                             1360       5           5       5                                              1436       20          50      80                                             1437                           10                                             FX 3                           5                                              ______________________________________                                    

As seen from the above table, compound 1436 inhibited blasttransformation most potently, with greater than 75% inhibition observedat 10 μg/ml. Some effect was observed for squalamine at thisconcentration, but the other steroids were considerably less active. Byusing this simple assay, compound 1436 was identified for use in thetreatment of T-cell lympho-proliferative diseases, including viralinfections which actively propagate on these cells.

Assays of similar design, employing a cell of interest and anappropriate growth factor, can be readily constructed. Thus, todetermine which steroid might be most useful in inhibiting proliferationof vascular smooth muscle cells following angioplasty, one need only setup a culture with human coronary smooth muscle and determine whichsteroid inhibits the PDGF-stimulated growth of these cells, as discussedbelow.

The Inhibition of a Spectrum of Cells

Using the assay of Baker et al., Cancer Res. 53, 1993,3052-3057,compound 1436 was observed to inhibit the growth of a verybroad spectrum of cells. As set forth in Table 7 below, all malignanttumors evaluated in tissue culture, endothelial cells and vascularsmooth muscle cells were sensitive to inhibition. Thus, compound 1436has application in the control of growth factor dependent proliferationof many types of tissue.

                  TABLE 7                                                         ______________________________________                                        CANCER CELLS INHIBITED IN VITRO BY COMPOUND                                   1436 (10 μg/ml):                                                           Human colon carcinoma  SW948                                                  Human colon carcinoma  HT29                                                   Human ovarian carcinoma                                                                              SKOV3                                                  Human melanoma         WM 1617                                                Lewis lung carcinoma                                                          Murine B16 melanoma                                                           Murine L1210 leukemia                                                         NON-TRANSFORMED CELLS INHIBITED IN VITRO BY                                   COMPOUND 1436 (10 μg/ml):                                                  Bovine pulmonary endothelial cells                                            Human microvascular endothelial cells                                         Human umbilical venous endothelial cells                                      Human coronary artery smooth muscle cells                                     ______________________________________                                    

Inhibition of NEE3

Compound 1436 was also found to inhibit rabbit NHE3. PS120 fibroblaststransfected with rabbit NHE3 were grown and acid preloaded by exposureto 40 mM NH₄ Cl as described above in conjunction with FIGS. 1A and 1B.Internal cellular pH changes expressed as the rate of pH recovery as afunction of restored extracellular sodium ion concentration followingexposure to 10 μg/ml of the compound were assayed with the fluorescentdye BCECF-AM as described above. The results are presented in FIG. 10.

Thus, compound 1436 is an inhibitor of NHE3. The inhibition of NHE3caused by compound 1436, however, does not adequately explain the verydifferent pharmacologic effects of squalamine and compound 1436 whenassessed on cells in culture and several in vivo models, as describedabove. This suggested that compound 1436 was inhibiting another NHE inaddition to NHE3. A reasonable candidate NHE is NHE5 (recently cloned,see Klanke et al., Genomics 25, 1995, 615-622), which is expressed atleast in lymphoid cells, brain, and testes.

Inhibition of NHE5-Expressing Cells

To determine whether NHE5 was the other NHE affected by compound 1436, atest was performed to evaluate whether cells inhibited by this compoundexpressed NHE5. Using the method of Klanke et al. and appropriate PCRprimers as described in Klanke et al., it was found that NHE5 wasexpressed in all cell types which exhibit sensitivity to compound 1436(see table below). Total cDNA was prepared from isolated total RNA ortotal RNA or polyA+ RNA purchased from Clontech Laboratories (Palo Alto,Calif.). Initial PCR cycle reactions were carried out as described inconjunction with Table 2 above, with primers specific for human NHE1,human NHE3 or human NHE5 with 80 ng cDNA or, in the case of polyA⁺ RNAas the cDNA source, with 1.5 ng cDNA. The annealing temperatures were57° C. in all instances. Hemi-nested PCR reactions were then carried outfor human NHE1 and NHE5 and nested PCR reactions for human NHE3 insecond-cycle PCR reactions, with the conditions as described above inconjunction with Table 2, except that the annealing temperature for thesecond PCR round for the primers to detect NHE5 was 65°0 C. Results aretabulated below.

                  TABLE 8                                                         ______________________________________                                        Antiporter:    NHE1     NHE3       NHE5                                       Rounds of PCR: 1      2     1    2     1    2                                 ______________________________________                                        Assayed cell line                                                             or tissue                                                                     adrenal gland  +      +     -    +     +    +                                 brain, whole   +      +     +*   +     +    +                                 small intestine                                                                              -      +     -    +                                            skeletal muscle                                                                              +      +     -    -/+*  -    +                                 HPAEC (endothelial)                                                                          +      +     +*   +     +    +                                 HMVEC (endothelial)                                                                          +      +     -    +     +    +                                 CaCO.sub.2 (epithetial)                                                                      +      +     +*   +     +    +                                 melanoma (WM1617)                                                                            +      +     +*   +     +    +                                 colon carcinoma                                                                              +      +     +*   +     -    -                                 (polyA.sup.+  RNA)                                                            leukemia HL-60 +      +     -    +     -    +                                 leukemia MOLT4 +      +     -    +     -    +                                 (polyA.sup.+  RNA)                                                            astrocytoma    +            +          +                                      glioblastoma   +            +          +                                      ______________________________________                                         Note:                                                                         *= multiple PCR bands observed.                                          

It is believed that NHE5, which is similar in sequence to NHE3, is themore effectively inhibited target of compound 1436. Cells which exhibitboth NHE3 and NHE5 would experience both NHE isoforms shut down bycompound 1436, but only NHE3 would be inhibited in the presence ofsqualamine.

Inhibition of Mouse Leukemia

Because of its inhibitory activity on the growth of numerous cancercells, compound 1436 was evaluated in a classical mouse model ofleukemia (Baker et al., Cancer Res. 53, 1993, 3052-3057). C57B mice wereinoculated with L1210 lymphoblastic leukemia cells at an inoculum thatcauses leukemia in 100% of animals. Mice received compound 1436 at 1, 5,10 mg/kg every 3 days intraperitoneally. As shown in FIG. 11,significant prolongation of life was achieved with the highest dose ofcompound 1436.

Of particular interest is the hematological profile determined duringthe course of treatment. Animals were treated with cisplatin andcompound 1436. As apparent from Table 9 below, animals treated withcisplatin developed a profound anemia by day 28, due to a suppression ofmarrow erythroid precursors. In contrast, animals treated with compound1436 exhibited a near normal hematocrit, with evidence of a robustgranulocyte count.

                  TABLE 9                                                         ______________________________________                                                      RBC (10.sup.6 /mm.sup.3)                                                                WBC (10.sup.3 /mm.sup.3)                                                  Early   Late  Early  Late                                 Agent   Treatment   Time    Time  Time   Time                                 ______________________________________                                        Cisplatin                                                                             Inoculate mice                                                                            9.4     1.5   8.1    18.1                                         5 × 10.sup.5 L1210                                                      cells ip, d 1                                                                 inject cisplatin                                                              8 mg/kg ip                                                            Cmpd. 1436                                                                            Inoculate mice                                                                            4.8     8.8   3.3     3.7                                         5 × 10.sup.5 L1210                                                      cells ip, inject                                                              compound 1436                                                                 10 mg/kg ip q4d                                                       ______________________________________                                    

Synergistic Inhibition of Tumor Growth

Based on the idea that tumor growth involves both the clonal expansionof a malignant cell along with the development of a supporting vascularsupply, a combination of compound 1436 with squalamine was tested todetermine whether it would achieve a synergistic effect on solid tumorgrowth. This concept was evaluated in the B16 melanoma model.

Animals were implanted with B16 melanoma followed by treatment withcompound 1436 or 1256 administered in a combined schedule or separately.As apparent from FIG. 12, when squalamine was administered at 5mg/kg/day or compound 1436 was administered at 10 mg/kg/every 3 days, nosignificant impact on tumor volume was observed. In contrast, when bothagents were administered together, a significant reduction in tumorgrowth was noted. Neither administration of squalamine at 15 mg/kg/daynor compound 1436 alone in a tolerable schedule could achieve thiseffect. Thus, a combination of these two compounds achieves a theraputicbenefit in tumors dependent on neovascularization that may preventmetastatic spread.

Effect of Aminosterols on Lymphotropic Viruses

Since compound 1436 inhibits the PHA-stimulated proliferation of human Tcells and controls the proliferation of a lymphoblastic leukemia in micewithout unfavorable toxicity as shown above, it seemed a reasonablecandidate for evaluation in vitro as an inhibitor of HIV. PHA-stimulatedlymphocytes were inoculated with a clinical isolate of HIV at amultiplicity of infection of 10. Fresh lymphocytes were obtained andstimulated with PHA and IL-2. After 3 days, 1000 TCID of virus-(HIVclinical isolate) were applied for 1 hour and there was a M.O.I. of1:10. The cells were washed and in a dose response fashion, and drug inmedia was applied. After 3 days, the supernatant was exchanged with 1/2volume of resh media and 1/2 the volume of fresh drug. After 7 days,detergent was added, and HIV P-24 Antigen was determined by Elisa.viability of the lymphocytes was evaluated along with appearance of theviral gene product p24. Results are tabulated below.

                  TABLE 10                                                        ______________________________________                                        INHIBITION OF HIV REPLICATION BY COMPOUND 1436                                Conc. μM   P-24 Elisa                                                                             % Viability                                            ______________________________________                                        0.5           40561    91                                                     1             7464     --                                                     5             3426     --                                                     10            421      95                                                     20            9        90.1                                                   ______________________________________                                    

As seen above, at 10 μg/ml compound 1436 effectively inhibited antigensynthesis by 97%, while retaining lymphocyte viability to 95%.

The above experiments clearly support the utility of compound 1436 inthe treatment of lymphotropic viral diseases. Based on these studies,the identification of the specific NHE inhibitors of specific cellulartargets of specific virus should permit the rational development of aneffective antiviral therapy for a given infectious agent. Thus, the NHEinhibitor from the aminosterols that acts on the respiratory epithelialcell should be effective in the treatment of respiratory viruses whichpropagate on these cells, such as Herpes, influenza and RSV. The conceptcan be generalized to viruses infecting the CNS (herpes) and liver(hepatitis). The approach prevents infection by the virus of thecellular target by preventing activation of the cellular NHE, requiredfor cellular proliferation and effective intracellular viralmultiplication.

Effect on Insulin Secretion

In studying additional roles for the aminosterols of this invention, itwas noted that the release of insulin from the islet cell of thepancreas requires activation of the islet cell's NHE, ultimatelyactivated through a mechanism triggered by glucose. It is believed thatoverstimulation of the islet cell might play a role in the depletion ofislet cell function in Type II disease. In addition, suggestions havebeen presented that genetic mechanisms leading to hyperactivity of theislet cell NHE may play a role in Type I disease.

Thus, the onset of diabetes in individuals genetically susceptible, orplaced into conditions of risk through acquired processes (obesity),might be delayed or allayed if islet cell function could be dampened.Inhibition of the NHE responsible for secretion of insulin could providetherapeutic benefit in these settings.

To study the effect of steroid administrator or the NHE responsible forsecretion of insulin, several of the aminosterols from shark liver wereadministered to fasting mice. Male CD-1 mice were assigned to one offour treatment groups. Whole blood glucose was tested using glucometer(Lifescan Glucometer II and One Touch test strips). Statistical analysiswas via one-way analysis of variance (ANOVA) followed by subsequentBonferonni's t-test. Results are tabulated below.

                  TABLE 11                                                        ______________________________________                                        EFFECT OF COMPOUND ON FASTING BLOOD GLUCOSE IN MICE                                                Total            Fasting Blood                           Dose        Com-     Dose             Glucose Mean                            Group n     pound    mg/kg Treatment  ± SEM (mg/dl)                        ______________________________________                                        1     5     --       --    Overnight fast,                                                                          38 ± 5.2                                                        blood obtained                                     2     4     1437     20    10 mg/kg i.v.                                                                            82 ± 15.3                                        (in            Dy 0 PM, overnight                                             H.sub.2 O)     fast, 10 mg/kg iv.                                                            Dy 1 AM, blood                                                                obtained 30 min.                                                              after 2d dose                                      3     4     1256     20    10 mg/kg i.v.                                                                            65 ± 7.3                                         (in            Dy 0 PM, overnight                                             H.sub.2 O)     fast, 10 mg/kg iv.                                                            Dy 1 AM, blood                                                                obtained 30 min.                                                              after 2d dose                                      4     3     1436     20    10 mg/kg i.v.                                                                            105 ± 8.0                                        (in            Dy 0 PM, overnight                                             D5W)           fast, 10 mg/kg iv.                                                            Dy 1 AM, blood                                                                obtained 30 min.                                                              after 2d dose                                      ______________________________________                                    

As apparent from the data above, blood glucose levels were elevatedbetween 2-3 fold normal after administration of these steroids. Thefasting blood glucose of Group 2 was significantly elevated compared toGroup 1 (p<0.05). The fasting blood glucose of Group 4 was significantlyelevated compared to Group 1. Thus, it appears that the intravenousadministration of compound 1436 in D5W (5% glucose) or compound 1437 inwater caused hyperglycemia in mice. It is assumed that the observedhyperglycemic response results from inhibition of insulin secretion, assuggested from basic physiological principles. Thus, the long-termchronic administration of a compound such as compound 1436 may be ofvalue in preventing or delaying the onset of both Type I and Type IIdiabetes.

Effect on Growth of Arterial Smooth Muscle

Aminosterols of the invention may also have utility in inhibiting thegrowth factor mediated proliferation of smooth muscle within the artery.Following coronary angioplasty, reocclusion commonly occurs, secondaryto reparative proliferation of the vascular smooth muscle within thewall of the surgically manipulated blood vessel. This process generallytakes place over the course of 7-10 days. To evaluate whether an agentcould prevent the growth factor mediated proliferation of smooth musclewithin the artery, compound 1436 was evaluated in vitro for its effecton the proliferation of human coronary artery smooth muscle. Results forcompound 1436 are shown in FIG. 13, and those for squalamine are shownin FIG. 14, with FIG. 15 being a composite logarithmic plot.

As seen from FIG. 13, at about 10-12 μg/ml compound 1436 was effectivein suppressing growth of these cells. For example, cells could bemaintained in a quiescent state in the presence of this steroid at about11 μg/ml without loss of viability. This experiment suggests that, forseveral days following angioplasty, local administration of compound1436 to the site of angioplasty via slow-release administration in aproximal vascular placement could reduce muscle proliferation during theperiod over which the vessel's endothelium reestablishes continuity andthe cellular events surrounding acute injury have subsided.

Effect on Growth and Weight Gain

During evaluation of the physiological effects of compound 1436 innormal growing mice, it became evident that this steroid suppresses bothlinear growth and weight gain in growing mice in a dose-dependentfashion. Animals were dosed QTD (i.p.) starting on Day 1. FIG. 16 showsthat C57B mice treated with 10 mg/kg, QTD i.p., 5 mg/kg QTD i.p., and 1mg/kg QTD i.p. exhibited a dose dependent reduction in growth. After 6doses, growth of the animals receiving 10 mg/kg QTD had been suppressedto a degree that growth was almost completely inhibited over about 1month from the initiation of treatment. Animals receiving 5 mg/kg QTDexperienced about a 50% reduction in growth, compared to untreatedcontrols, while animals receiving 1 mg/kg QTD were affected by about10%. Striking was the apparent health of the treated animals--all wereactive, normally proportioned, not-cachectic, and in excellent apparentclinical health. They appeared very much like hypophysectamized animalsmight appear.

Compound 1436 clearly inhibits the growth of many different types ofcell and tissue and this, to some extent, explains the profoundsuppression of growth observed. However, the extraordinary good healthof these animals suggests that an additional mechanism must beinvolved--one involving inhibition of pituitary function. Compound 1436is believed to partially inhibit secretion of anterior pituitaryhormones, resulting in the observed growth suppression.

This property of compound 1436, regardless of its precise mechanism,suggests that it can produce an unprecedented form of antiproliferativeeffect when administered to an animal. It will not only inhibitgrowth-factor induced cellular proliferation by acting on theproliferating cell, but also inhibit growth-promoting hormone secretionat a central, endocrine level. Thus, compound 1436 places the animal ina "growth-inhibited" state. In such a state, malignant cells will notreceive optimal exogeneous hormonal stimulation from hormones such asgrowth hormone, and perhaps LH and FSH. Secretion of hormones such asestrogen and progesterone, as well as insulin, are likely to bedysregulated. Virally infected cells will be placed underphysiologically unfavorable conditions, and the efficiency of viralinfection should be dramatically reduced. Immunologically foreign cells,suppressed in growth, should be cleared by existing immune systems, nowgiving a chance to catch up kinetically to these "foreign" cells.

Effect on Arterial Resistance

Compound 1436 has also been found to reduce arterial resistance in therat after intravenous (i.v.) administration. A 200-g rat wascatheterized in the right carotid artery, and the compound wasintroduced over a ten-second period to a total dosage of 5 mg/kg. Within30 seconds, the mean arterial pressure had fallen from about 100 mm Hgto about 70 mm Hg, with a marked reduction in pulse pressure from about40 mm to about 10 mm. Despite the fall in blood pressure, no significantincrease in heart rate was observed. If cardiac output was basicallyunaffected, reduction in blood pressure would have resulted principallyfrom a reduction in system resistance.

The effect was followed for 30 minutes, without significant change. Atthat time, 40 μg of noradrenaline was introduced. An almost immediateincrease in blood pressure was observed, with an associated increase inpulse pressure. This data demonstrate that the effect of compound 1436is readily reversible by standard pharmacological practice.

The ability of compound 1436 to reduce arterial resistance and arterialblood pressure indicates its application as an antihypertensive agent.Because it does not appear to induce a compensatory tachycardia, the neteffect is to reduce cardiac afterload. A physiological consequence ofthis type of cardiovascular effect would be to slow the process ofcardiac hypertroph and arteriolar smooth muscle proliferation. Becauseof these properties, compound 1436 should be an effective treatment ofcongestive heart failure, where reduction in afterload would be desired.Its rapidity of action and ready reversibility, along with minimaltachycardic effect, make the compound a valuable therapeutic agent.

Thus, compound 1436 represents an antiproliferative and therapeuticagent with previously unknown and valuable properties and utilities. Itclearly can be utilized in disorders where suppression of growth ofspecific tissues or entire organ systems is desired.

Suppression of Cardiotoxic Effects of Ischemia

It has been suggested that inhibitors of the NHE family could play atherapeutic role in the treatment of cardiac ischemic states. Thesestates occur after heart attacks, during heart failure, and in thecourse of transplantation of an organ from donor to recipient.

To determine if compound 1436 has such utility, the following experimentwas performed. The heart of a juvenile Xenopus laevis frog was dissectedfrom the living animal. The heart was placed into a Petri dishcontaining Krebs-Ringer buffer with adrenaline 50 μg/ml, and examinedwith the naked eye. At room temperature, the heart continued beating ina coordinated fashion (atrial beat followed by ventricular beat) forabout one hour. In the presence of 10 μg/ml of compound 1436,spontaneous beating persisted up to 24 hours. The atrial pacemaker andthe conduction of the atrial beat to the ventricle remained vigorousover this period.

Although the precise mechanism explaining this phenomenon of persistenceof cardiac activity ex vivo is not fully understood, it is believed thatcompound 1436, by inhibiting NHE3 and NHE5, prevents accumulation ofintracardiac calcium by blocking these NHEs. It is the currentunderstanding in the art that intracellular acid accumulating duringischemia is exchanged by the NHE for extracellular sodium. In turn, thesodium driven into the cell is subsequently excreted in exchange forextracellular calcium through the action of a sodium/calcium exchanger.It is the calcium entering via this route that leads to cardiac deathand cardiac arrhythmia. By blocking the NHE, compound 1436 preventsprotons or acid from leaving the cardiac cell, reducing energyconsumption and work output, effects which are protective to the cell,along with preventing the ultimate entry of damaging calcium.

Anti-Proliferative Assays and Tumor Growth Suppression Assays asCharacterizing Assays

As above, the tadpole assay was used to screen for additional compounds.In the presence of 10 μg/ml of compound 1436, the stage 59-60 Xenopustadpole experiences dramatic disruption of melanocytes over its head,trunk, and tail, along with depression of its nervous system. No effectsare observed on epithelial cell integrity, vascular flow, erythrocytevolume, tissue integrity, muscle fiber striation, or GI tract activity.

Using the tadpole assay to screen for functionally similar compounds,compounds 353, 371 and 413 were found to produce effects like thoseproduced by compound 1436. Of these, compound 353 is especiallypreferred because of its ease of synthesis as described above. Thiscompound was also found to exhibit other advantageous properties.

Using the growth suppression methods set forth above, it was determinedthat compound 353 exhibits potent activity against melanoma and a numberof cancer cells as set forth below:

                  TABLE 12                                                        ______________________________________                                        CYTOTOXIC ACTIVITY OF COMPOUND 353 AGAINST                                    CANCER CELLS                                                                  Cell               IC.sub.50 (μg/ml)                                       ______________________________________                                        Human melanoma WM 1167                                                                           3.0                                                        Lewis Lung carcinoma                                                                             1.9                                                        ______________________________________                                    

In addition, using the method of Baker et al., Cancer Res. 53, 1994,3052-3057, it was observed that the effect of compound 353 on the growthof melanoma is most pronounced over 48 hours, with less of an effectnoted within the first 12 hours of incubation, as shown in FIG. 17A. Forcomparative purposes, the effect of squalamine in human melanoma isshown in FIG. 17B.

Analysis of the cells exposed to compound 353 reveals that apoptoticcell death has been induced. Thus, this aminosterol exhibits the samehighly selective mechanism of killing as does compound 1436.

Although as set forth above, compound 1436 exhibits melanocytedisruptive activity in the tadpole, it causes vitelline capillaryregression with about the same potency as squalamine. In contrast,compound 353 exhibits no effect on the chick embryo capillary bed. Thus,it appears that compound 353 inhibits NHE5 to a greater extent thanNHE3, with even greater selectivity than compound 1436. Compound 353demonstrates that it is possible to create aminosterols that exhibitgreater specificity than naturally occurring molecules.

Compound 1437 (Fraction 4) contains an unusual ergosterol-like sidechain. This molecule can be distinguished readily from all othersteroids extracted from shark on the basis of its dramatic effect on theembryonic epithelium covering the tadpole tale.

Using the tadpole assay described above, within 60 minutes of exposureto this steroid at 10 μg/ml, the larval skin was observed to shed off ina sheet. The speeded appearance of the process suggests that an NHEexpressed by this epithelial tissue is the target. Since NHE activityand cell membrane proteins involved in adhesion cross-communicate(Schwartz et al., Proc. Nat'l. Acad. Sci. 888, 7849-7853), it isproposed that inhibition of NHE on the epithelium results in disruptionof cellular contacts between the epithelium and its substratum, leadingto a shedding effect.

Using the assay described above, the anticancer effects of compound 1437against several cancer lines was assessed. Compound 1437 was found toexhibit anticancer activity against the human ovarian carcinoma, SKOV3.Thus, compound 1437 should find use in the treatment of carcinomasexhibiting a sensitive phenotype.

As the study above demonstrates, compound 1437 targets a"mesothelium-like" epithelial layer, a skin layer that is comprised ofonly one cellular layer. Such a layer resembles epithelial surfaces suchas the human peritoneum, synovium, pericardium and ependyma.Accordingly, compound 1437 should exhibit antiproliferative effects onthese tissues and malignancies which derive from them. In addition,these tissues can support viral infections, and therefore in theseinstances the compound should provide therapeutic antiviral benefit.

By use of the Xenopus tadpole assay, it is possible to identifycompounds that exhibit little chemical resemblance to compound 1437, butproduce the same pharmacological effect with respect to epithelialshedding. Using such a method, it was found that compounds 409, 410,416, 431, 432 and 433 are functionally similar to compound 1437.

Steroid 1360 (Fraction 2) contains a side chain bearing a keto group oncarbon 24 and a sulfate on the C 27 hydroxyl. Although somewhat similarin structure to squalamine, it exhibits a dramatically differentpharmacologic profile in both the tadpole and chick embryo assays.

When stage 59-60 tadpoles were introduced into a 10 μg/ml solution ofcompound 1360, extensive vasocclusion occurred within 60 minutesthroughout the tail--the distal portions to a greater extent than theproximal. Occlusion occurred due to the visible swelling of erythrocytesfollowed by rupture and release of nuclei. Nuclei were shuttled by thedesign of the vascular bed into distal arteries and veins, which can beanalogized to a coin separating machine separating coins of differentsize and weight into specific collecting tubes. As the nuclei pooledwithin these vessels, blood flow stopped proximally. Within 20-30minutes after nuclear plugs formed, tissue surrounding these plugs beganto break down. It appeared as if the nuclei were releasing hydrolyticenzymes that were essentially dissolving the ground substance holdingthese tissues.

In the chick embryo assay, application of compound 1360 produced adifferent effect than seen in the tadpole. Within 20 minutes, the bloodcirculating through the embryonic vessels exhibited a brighter redcolor, reflecting a higher degree of oxygenation than the red cells notexposed to the compound. Although many numerous mechanisms might explainthis effect, it is believed that the red cell of the chick isexperiencing a more alkaline internal pH after exposure to compound1360. This could arise through activation of the NHE of this cell.Furthermore, activation of the exchanger would also cause cellularswelling--a phenomenon observed in tadpole.

It is known that the nucleated erythrocytes of amphibia and fish (andprobably birds) express a specific NHE, termed NHEl-beta. Unlike allothers characterized, this exchanger is activated by cAMP and isinfluenced by the state of hemoglobin oxygenation. This data suggestthat compound 1360 will be shown to activate this exchanger.Furthermore, the chemical structure of the compound makes it ideallysuited to function in the suggested fashion. It has been discoveredthat, under slightly alkaline conditions, compound 1360 undergoes adehydration of the 27 hydroxyl, loss of sulfate, and generation of thecorresponding 27-ene, as set forth in the scheme below: ##STR24##

Thus, as the alkalinity of the interior of a cell increases, thelifetime of compound 1360 decreases, thereby providing an extraordinaryform of "feedback." It is possible that compound 1361, the product ofthis hydrolytic conversion, is inhibitory to the same NHE.

The data demonstrate that compound 1360 clearly interacts with a NHEpresent on embryonic stage blood cells. Since the human fetus generatesnucleated red cells comparable in size to those of the birds, fish, andamphibia, it is thought that certain human blood cells, perhaps fetal,will also represent cellular targets of this compound. Activation of thefetal NHE might find use in strategies designed to protect the fetusfrom hypoxic damage.

The full scope of applications for compound 1360 awaits description ofthe distribution of the erythrocyte NHE isoform in man. However, itappears to be stimulatory activity in some settings, rather than aninhibitor of an NHE. In any event, compound 1360 could be used for thefollowing: antibacterial, antifungal, antiviral, etc.; fetal distresstreatment; and hematologic malignancies treatment.

Although the chemical structure of Fraction 3 (FX 3) is yet to be fullydetermined, from its thin layer chromatographic properties it has aspermine associated with the steroid, much like compound 1436. Thissteroid has a profound effect on the Embryonic skeletal muscles of thetadpole.

In the tadpole assay, within 1 hour after exposure, leakage of brownpigment from the tail muscle bundles of stage 59-60 tadpole wasobserved. Cross striations became fuzzy and obscure. Heartbeat and otherfunctions, including muscle activity in the limbs, were unaffected.These observations suggest that FX 3 is targeting primitive mesenchyme,including muscle.

If the observations of the tadpole extend to man, then FX 3 shouldprofoundly affect the proliferation of certain mesenchymal cells. Thus,it would have use in the treatment of a variety of cancers ofmesenchymal origin, such as cancers of striated muscle, cartilage,fibroblastic tissues, bone, and fatty tissue.

In addition, if proliferation of fibroblasts is affected, then FX 3would have application in the control of fibroblastic proliferation insettings where this process is unwanted. Thus, scarring after CNS injurymight be prevented. Unwanted scarring after surgery at sites complicatedby fibrosis would be serious therapeutic targets. Generalized conditionsof fibroblastic proliferation, such as seen in heart, kidney and liverdisease, might be allayed.

If proliferation of muscle is inhibited, FX 3 could find use in theinhibition of hyperplastic diseases of muscle, such as in certain formsof cardiac disease.

Through use of the Xenopus tadpole assay, several aminosterols have beenidentified as exhibiting pharmacologic activity similar to that seen forFX 3. These compounds include compounds 370, 412, 458, 459, 465 and 466.These compounds in general share the spermine moiety. They are simplerchemically than squalamine and offer a less expensive route to drugdesign than the naturally occurring steroids.

The structure of the Fraction 1 (FX 1A) steroid is shown above. Itappears to undergo conversion to another molecule (FX 1B) rapidly inwater. FX 1A exerts a distinctive pharmacologic effect on the Xenopustadpole using the assay set forth above.

Within several hours of introduction of this steroid into the watersurrounding the tadpole, fecal elimination is dramatically increased.Since the GI tract of a number of vertebrates utilizes NHE in thecontrol of gut fluid secretion, it is believed that Fraction 1 acts onsuch an NHE. The increase in fecal material could correspond to"diarrhea," a condition which occurs in man when the colonic NHE isinhibited. Since this steroid has little effect on overall activity,muscle integrity or viability of any visible tissue, it might serve aphysiological function such as regulation of sodium-water exchange.

Although the uses will be clearer after the steroid and target arebetter characterized, the tadpole data suggest that Fraction 1 will finduse in the modulation of sodium/proton exchange in certain physiologicalderangements. These include treatment of hypertension, cystic fibrosisand constipation.

Because of its effects on bowel fluid dynamics, this agent may be as aantimicrobial--one which would effect killing of susceptible bacteria,parasites, fungi, etc., while promoting the discharge of the infectiousload from the gut. Fraction 1 may also find use as an effectiveantibacterial, antiparasitic or antifungal agent.

Through the use of the Xenopus tadpole assay, aminosterols withpharmacologic activity similar to Fraction 1 have been identified.Surprisingly, these compounds have been found to include compound 1363and compound 414. Although these compounds exhibit potencies comparableto that of Fraction 1, they have chemically simpler structures.

Preliminary data has revealed the presence of a least two hydrophilicsteroids eluting slightly ahead of Fraction 1 on the reversed-phaseseparation depicted in FIG. 9. The structures of these steroids arepresented below. ##STR25##

FX1C and FX1D are seen to be steroids with a single sulfate likesqualamine and an additional hydroxyl, resembling compound 1437.

Additional Aminosterol Structures

From the diverse aminosteroids isolated from Squalus acanthias, it ispossible to predict the existence of related aminosterols not as yetisolated from this animal's tissues. These sterols can be deduced toexist in vertebrate tissues based on the structures determined to dateand the known biochemical transformations the cholesterol side chain canundergo (Tammar, "Bile Salts in Fishes," Chemical Zoology, (eds. Florkinet al.), Academic Press, 1974, 595-612).

Thus, based on the existence of squalamine, which bears a 24 sulfatedhydroxyl, one should be able to find other derivatives with thesqualamine steroidal nucleus and aminosterol portion, but differing inthe position of the sulfated hydroxyl on the side chain as shown below.Since hydroxylation can occur on carbons 25, 26 or 27, and since eachwould represent a stereospecific chemical entity, it is reasonable toexpect their existence in nature and to assume they would exhibitdistinct pharmacological properties.

Similarly, the existence of steroids bearing a single sulfate along witha second hydroxyl in the cholesterol side chain suggests potentialdiversity in the pattern of side-chain sulfation and single-sitehydroxylation. Thus, aminosterols likely exist in nature where sulfationis found on carbons 24, 25, 26 or 27. In turn, each of these foursulfated aminosterols can be hydroxylated at available carbons 24, 25,26 and 27. At least the following steroids may be isolated from naturalproducts, based on inductive logic and the data revealed herein:##STR26##

Structure-Activity Considerations for NHE Inhibitors

Based on the information given above, key structural elements of theaminosterol inhibitors of the sodium/proton exchangers can now bededuced. The key core structure contains a steroid nucleus and adistinctive side chain. An aminosterol portion specifies interaction ofthe molecule with a NHE. The side chain, bearing free or sulfatedhydroxyl groups, determines the specificity for a specific NHE isoform.In addition, the presence of spermine or spermidine attached to thesteroid extends the spectrum of activity. Based on this generalizationit can be readily seen that the structure of the side chain impartsgreat specificity.

Thus, other synthetic steroidal NHE inhibitors can be designed withgreat pharmacological specificity by considering the modular nature ofthe molecule. Chemical entities which mimic the aminosterol in shape andmolecular surface characteristics will interact with the NHE family.Such chemical mimics of the steroidal nuclei are known and widely usedin the synthesis of non-steroidal estrogen agonists and antagonists.Coupling of specific cholesterol side chains to these steroidomimeticstructures will in turn establish specificity for individual NHEisoforms.

Antimicrobial Activity

The aminosterol NHE inhibitors represent a class of antibiotics based onmechanism of action. Because these agents also interact with specificNHE isoforms in human tissues, prudent selection of an antibiotic ofthis class can eliminate undesirable side effects, due to host NHEinhibition, or potentiate the therapeutic intent. Thus, use of an agentlike compound 1436 would suppress lymphoid proliferation during activetreatment of an infection. Oral administration of Fraction 1 mayincrease bowel fluid transit as it kills parasitic targets. An effectiveantifungal agent can be designed further to increase specificity for itspathogenic target over sensitive vertebrate isoforms.

As seen in Table I at the end of this specification, theantibacterial/antifungal spectrum differs from compound to compound.Thus, it is possible to achieve an antimicrobial steroid with or withoutsqualamine-like pharmacological activity.

As set forth in Table II, which follows Table I, the activities ofnatural and synthetic aminosterols in the different assays vary. Inlight of the foregoing, it is now possible to screen for steroids withor without squalamine-like pharmacological activity.

Selection of NHE Isoform

Through the use of molecular biological techniques, it is possible todetermine which NHE isoforms are expressed in specific cells, such asmalignancies. Human melanoma expresses NHE1, NHE3 and NHE5, and humanadenocarcinoma expresses principally NHE3 (see Table 8).

Thus, treatment of this type of adenocarcinoma might most effectively beaccomplished with the use of a more specific NHE3 inhibitor, such assqualamine or compound 319. In contrast, melanoma expresses considerableamounts of NHE5 along with NHE3. Hence, treatment of this malignancyshould include an inhibitor of both NHE3 and NHE5, such as compound1436, alone or in combination with squalamine.

In summary, the invention allows for the utilities of the aminosterolNHE inhibitors to be established through diagnostic evaluation of theNHE isoforms expressed in the target tissues. Diagnostic approaches caninclude immunological detection of the specific NHE isoform protein or amolecular biological procedure such as PCR, utilizing specific sequenceinformation, and standard techniques.

Thus, other embodiments of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention. The embodiments and preferred features described aboveshould be considered as exemplary, with the invention being defined bythe appended claims.

                                      TABLE I                                     __________________________________________________________________________    ANTIBIOTIC ACTIVITY OF NATURAL AND SYNTHETIC AMINOSTEROLS                                                                 Antibiotic Activity                                                           MIC Values (μg/mL)                                                         S.   E.   P.   C.                 Structure                                   aureus                                                                             coli aerug                                                                              albicans           __________________________________________________________________________     ##STR27##                                   4   >256 >256  64                 ##STR28##                                   16  >256 >256 >256                ##STR29##                                   4-16                                                                               32  128   64                 ##STR30##                                   8-16                                                                               64  128   64                 ##STR31##                                   8   128-256                                                                            128  256                 ##STR32##                                  0.5-1                                                                              2-4   16   8                  ##STR33##                                  2-4  128  128  128                 ##STR34##                                   64  32-64                                                                               32   8                  ##STR35##                                  128   32   64  >256                ##STR36##                                   16  128   64   32                 ##STR37##                                   8   64-128                                                                              64  16-32               ##STR38##                                  16-32                                                                              128  256   64                 ##STR39##                                  2-4  4-8   16   16                 ##STR40##                                  4-8   32   64   32                 ##STR41##                                   32   64   32  128                 ##STR42##                                   16   64   32   32                 ##STR43##                                   2   >256 >256  32                 ##STR44##                                   4    64   64   32                 ##STR45##                                   4    32   64   64                 ##STR46##                                  2-4   64  128   16                 ##STR47##                                   16  32-64                                                                               16   32                 ##STR48##                                   4   32-64                                                                              256   64                 ##STR49##                                   16   64  128  128                 ##STR50##                                   64  256  >256 256                 ##STR51##                                   4   32-64                                                                               64   64                 ##STR52##                                   8   128  128   32                 ##STR53##                                   4    32  128   4                  ##STR54##                                   2   >256 >256  2                  ##STR55##                                   2   >256 >256  2                  ##STR56##                                   4    32   64   2                  ##STR57##                                   4    64   64   2                  ##STR58##                                   32   64  128   16                 ##STR59##                                   16   16   32   16                 ##STR60##                                   8    64   64   2                  ##STR61##                                   8    64   8    8                  ##STR62##                                   8   256   64   32                 ##STR63##                                   32   64   64   64                 ##STR64##                                  128  128  256  256                 ##STR65##                                   8    8   16-32                                                                               32                 ##STR66##                                  16-32                                                                               64  128   32                 ##STR67##                                   16  32-64                                                                              128   8                  ##STR68##                                   1    8-16                                                                               64  2-4                 ##STR69##                                  >256 >256 128  >256                ##STR70##                                                                     ##STR71##                                                                     ##STR72##                                   2    16   8    8                  ##STR73##                                   4    8-16                                                                               4    4                  ##STR74##                                   4    32   64   2                  ##STR75##                                  1-2   32   64   2                  ##STR76##                                  2-4   32  128   4                  ##STR77##                                   2    32   32   2                  ##STR78##                                   16   16   8    4                 __________________________________________________________________________

    TABLE II                                                                         - ACTIVITY OF NATURAL AND SYNTHETIC AMINOSTEROLS                               IN CHICK EMBRYO AND TADPOLE ASSAYS                                             Minimum Effective Concentration (μg/mL)                                     Chick                                                                          embryo   HM                                                                    diss.  Cord MTT ELC                                                            assay Tadpole (10 μg/mL) Formation assay MTT                                Structure (μg) V M E TB GI Mus Tox (μg/mL) (μg/mL) (μg/mL)         303 >10 -                                                                      ##STR79##                                                                     318 >10 - - - - - - -                                                           ##STR80##                                                                     319 0.001 + - - - - - - 13.8                                                    ##STR81##                                                                     353 >1 - + - - - - + 10 3.0 1.9                                                 ##STR82##                                                                     354 >10 - - - - - - -                                                           ##STR83##                                                                     355  + - - - - - ±                                                           ##STR84##                                                                     356  + - - - - - +                                                              ##STR85##                                                                     370 >10 - - - - - + +  40                                                       ##STR86##                                                                     371 1-10 - + - - - - +                                                          ##STR87##                                                                     364 1 + - ± - - - +                                                          ##STR88##                                                                     396 10 - - - - - - +                                                            ##STR89##                                                                     397 1 - - - - - - +                                                             ##STR90##                                                                     399 >10 - - - + - - -                                                           ##STR91##                                                                     409 >1 - - + + - - +                                                            ##STR92##                                                                     410 0.01 ± - + + - - + 10 2.6                                                ##STR93##                                                                     381 >10 - - - - - - +                                                           ##STR94##                                                                     382 0.01 - - - - - - +                                                          ##STR95##                                                                     393 >10 - ± - - - - +                                                        ##STR96##                                                                     394 >10 -                                                                       ##STR97##                                                                     395 >10 - - - - - - +                                                           ##STR98##                                                                     459 >1 - - ± - - + +  5.0                                                    ##STR99##                                                                     465  - - ± - - + +                                                           ##STR100##                                                                    466 1 - - ± - - + +                                                          ##STR101##                                                                    467  - - - - - - +                                                              ##STR102##                                                                    431 >1 ± - + + - - +                                                         ##STR103##                                                                    432 >1 - - + - - - +                                                            ##STR104##                                                                    433 1 - - + - ± - -                                                          ##STR105##                                                                    448 1 ± - + + - - +                                                          ##STR106##                                                                    449 >1 - - ± ± - - +                                                      ##STR107##                                                                    458 >1 - - ± - - + +  6.8                                                    ##STR108##                                                                    411 >1 ± - - - - - +                                                         ##STR109##                                                                    412 1 - - - - - + + >10 18.1                                                    ##STR110##                                                                    413 >1 - + - - - - + >10                                                        ##STR111##                                                                    414 >1 - - - - + - -                                                            ##STR112##                                                                    415 >1 - - - - - - - >10                                                        ##STR113##                                                                    416 >1 ± - + + - - +  2.4                                                    ##STR114##                                                                    417 >1 - - - - - - -                                                            ##STR115##                                                                    1256 0.01 + - - - - - + 0.01-0.1 7.8 13.2                                       ##STR116##                                                                    1360 >10 - - + + - - -                                                          ##STR117##                                                                    1363  - - - - + - -                                                             ##STR118##                                                                    1436 1 + + ± - - - +  6.9 16.7                                               ##STR119##                                                                    1437 >1 - - + - - - -                                                           ##STR120##                                                                    V = vascular,                                                                  M = melanocytes,                                                               E = epithelial,                                                                TB = tapoebeardoes                                                             GI = gastrointestinal,                                                         Mus = muscle,                                                                  Tox = lethality at 2 hrs                                                  

What is claimed is:
 1. A compound having the following structure:##STR121## or a pharmaceutically acceptable salt thereof.
 2. A compoundaccording to claim 1, wherein the compound is in a purified form.
 3. Acompound according to claim 1, wherein the compound is in an isolatedform.
 4. A compound according to claim 3, wherein the compound isisolated from shark liver.
 5. A composition comprising the compound ofclaim 1, and a pharmaceutically acceptable vehicle or carrier.
 6. Acompound having the following structure: ##STR122## or apharmaceutically acceptable salt thereof.
 7. A compound according toclaim 6, wherein the compound is in a purified form.
 8. A compoundaccording to claim 6, wherein the compound is in an isolated form.
 9. Acompound according to claim 8, wherein the compound is isolated fromshark liver.
 10. A composition comprising the compound of claim 6, and apharmaceutically acceptable vehicle or carrier.